US12280124B2 - Antibody-drug conjugates comprising GLP1 peptidomimetics and uses thereof - Google Patents

Antibody-drug conjugates comprising GLP1 peptidomimetics and uses thereof Download PDF

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US12280124B2
US12280124B2 US17/475,248 US202117475248A US12280124B2 US 12280124 B2 US12280124 B2 US 12280124B2 US 202117475248 A US202117475248 A US 202117475248A US 12280124 B2 US12280124 B2 US 12280124B2
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antibody
linker
compound
payload
glp1r
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US20220096648A1 (en
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Amy Han
Haruka Okamoto
William Olson
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Regeneron Pharmaceuticals Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics

Definitions

  • the present disclosure relates to protein-drug conjugates (e.g., antibody-drug conjugates), pharmaceutical compositions, and methods of treating disease therewith. Also provided are peptidomimetic payloads and linker-payloads and methods of making same. More specifically, the present disclosure relates to protein-drug conjugates (e.g., antibody-drug conjugates) comprising GLP1 peptidomimetics and methods of treating GLP1R-associated conditions therewith.
  • Diabetes is a chronic disease of abnormal glucose metabolism. 425 million people are estimated to be living with diabetes worldwide.
  • Global diabetes drugs include insulin, DPP-4 inhibitors, glucagon-like peptide 1 receptor (GLP1R) agonists, but most patients do not achieve combined treatment goal to manage hyperglycaemia and cardiovascular risk factors.
  • GLP1R glucagon-like peptide 1 receptor
  • GLP1R Glucagon-Like Peptide 1 Receptor
  • GLP1R is the receptor for glucagon-like peptide 1 (GLP1) and is expressed in the pancreatic beta cells. GLP1R is also expressed in the brain where it functions in the control of appetite, memory and learning. GLP1R is a member of the secretin family (Class B) of G protein-coupled receptors (GPCRs). Upon binding of its ligand, GLP1, GLP1R initiates a downstream signaling cascade through G ⁇ s G-proteins that raises intracellular cyclic AMP (cAMP) levels, which leads to the transcriptional regulation of genes (Donnelly 2011). Activation of GLP1R results in increased insulin synthesis and release of insulin.
  • cAMP cyclic AMP
  • GLP1R and GLP1 are highly validated targets for obesity and type 2 diabetes. Marketed GLP1R agonists increase insulin secretion, thereby lowering blood glucose levels, but they require weekly or more frequent administration.
  • the present disclosure meets the needs and provides other advantages.
  • BA is a Glucagon-like peptide-1 receptor (GLP1R)-targeting antibody or an antigen-binding fragment thereof.
  • GLP1R-targeting antibody is a GLP1R agonist antibody.
  • the GLP1R-targeting antibody is 5A10, 9A10, AB9433-1, h38C2, PA5-111834, NLS1205, MAB2814, EPR21819, or glutazumab.
  • the linker L is attached to one or both heavy chains of the BA. In some embodiments, the linker L is attached to one or both heavy chain variable domains of the BA. In some embodiments, the linker L is attached to one or both light chains of the BA. In some embodiments, the linker L is attached to one or both light chain variable domains of the BA.
  • the linker L is attached to BA via a glutamine residue.
  • the glutamine residue is introduced to the N-terminus of one or both heavy chains of the BA.
  • the glutamine residue is introduced to the N-terminus of one or both light chains of the BA.
  • the glutamine residue is naturally present in a CH2 or CH3 domain of the BA.
  • the glutamine residue is introduced to the BA by modifying one or more amino acids.
  • the glutamine residue is Q295 or N297Q.
  • the linker L is attached to BA via a lysine residue.
  • the antibody or antigen-binding fragment thereof is aglycosylated. In some embodiments, the antibody or antigen-binding fragment thereof is deglycosylated. In some embodiments, the antigen-binding fragment is an Fab fragment.
  • m is 1. In one embodiment, m is an integer from 2 to 4. In one embodiment, m is 2.
  • more than one L-P is attached to the BA. In some embodiments, two L-Ps are attached to the BA.
  • the linker L has the structure of formula (L′): —La—Y—Lp- (L′),
  • Y has a structure selected from the group consisting of:
  • Lp comprises a polyethylene glycol (PEG) segment having 1 to 36 —CH 2 CH 2 O— (EG) units.
  • the PEG segment comprises between 2 and 30 EG units.
  • the PEG segment comprises between 4 and 24 EG units.
  • the PEG segment comprises 4 EG units, or 8 EG units, or 12 EG units, or 24 EG units.
  • the PEG segment comprises 4 EG units.
  • the PEG segment comprises 8 EG units.
  • Y-Lp has a structure selected from the group consisting of:
  • the Lp comprises one or more amino acids selected from glycine, serine, glutamic acid, alanine, valine, and proline and combinations thereof. In some embodiments, the Lp comprises 1 to 10 glycines. In some embodiments, the Lp comprises 1 to 6 serines. In some embodiments, the Lp comprises 1 to 10 glycines and 1 to 6 serines. In some embodiments, the Lp comprises 4 glycines and 1 serine.
  • the Lp is selected from the group consisting of Gly-Gly-Gly-Gly-Ser (G 4 S) (SEQ ID NO: 1), Ser-Gly-Gly-Gly-Gly (SG 4 ) (SEQ ID NO: 2), and Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (G 4 S-G 4 S) (SEQ ID NO: 3).
  • the Lp comprises a combination of a PEG segment having 1 to 36 EG units and one or more amino acids selected from glycine, serine, glutamic acid, alanine, valine, and proline and combinations thereof.
  • the serine residue comprises a carbohydrate group.
  • the serine residue comprises a glucose group.
  • Lp has a structure selected from the group consisting of:
  • Y-Lp has a structure selected from the group consisting of: (SEQ ID NOS 31-36, respectively, in order of appearance)
  • La comprises a polyethylene glycol (PEG) segment having 1 to 36 —CH 2 CH 2 O— (EG) units. In some embodiments, the PEG segment comprises 4 EG units, or 8 EG units, or 12 EG units, or 24 EG units. In some embodiments, the PEG segment comprises 8 EG units. In some embodiments, La has a structure selected from the group consisting of
  • La comprises one or more amino acids selected from glycine, threonine, serine, glutamine, glutamic acid, alanine, valine, leucine, and proline and combinations thereof. In some embodiments, La comprises 1 to 10 glycines and 1 to 6 serines. In some embodiments, La comprises 4 glycines and 1 serine.
  • La is selected from the group consisting of Gly-Gly-Gly-Gly-Ser (G 4 S) (SEQ ID NO: 1), Ser-Gly-Gly-Gly-Gly (SG 4 ) (SEQ ID NO: 2), Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly (G 2 S-G 2 S-G 2 ) (SEQ ID NO: 25), and Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Gly (G 4 S-G 4 ) (SEQ ID NO: 26).
  • La comprises a combination of a PEG segment having 1 to 36 EG units and one or more amino acids selected from glycine, threonine, serine, glutamine, glutamic acid, alanine, valine, leucine, and proline and combinations thereof.
  • La is selected from the group consisting of (SEQ ID NOS 37-38, respectively, in order of appearance):
  • La comprises a —(CH 2 ) 2-24 — chain. In some embodiments, La comprises a combination of a —(CH 2 ) 2-24 — chain, a PEG segment having 1 to 36 EG units and one or more amino acids selected from glycine, threonine, serine, glutamine, glutamic acid, alanine, valine, leucine, and proline and combinations thereof. La is selected from the group consisting of (SEQ ID NOS 39-40, respectively, in order of appearance):
  • P has the structure
  • X 3 is selected from —(CH 2 ) 2-6 —NH—and —(CH 2 ) 2-6 —Tr-, where Tr is a triazole moiety; n is 1, and X 4 is H.
  • X 1 is
  • X 3 is —(CH 2 ) 2-6 —NH—; n is 1; X 4 is H, and X 5 is selected from —OH, —NH 2 , —NH—OH, and
  • X 1 is
  • X 3 is —(CH 2 ) 2-6 —NH—; n is 1, and X 4 is H.
  • X 1 is
  • X 3 is —(CH 2 ) 2-6 —NH—; n is 1; X 4 is H; X 6 is independently at each occurrence selected from H and —CH 2 OH, and X 7 is H.
  • X 1 is
  • X 3 is —(CH 2 ) 2-6 —Tr-, where Tr is a triazole moiety; n is 1; X 4 is H, and X 5 is
  • X 1 is
  • X 3 is —(CH 2 ) 2-6 —NH—; n is 1; X 4 is H; X 6 is independently at each occurrence selected from H and —CH 3 ; X 7 is
  • X 8 is —NH 2 .
  • X 1 is
  • X 3 is —(CH 2 ) 2-6 —NH—; n is 1; X 4 is H, and X 3 is H.
  • X 1 is
  • X 3 is —(CH 2 ) 2-6 —NH—; n is 1; X 4 is H; X 6 is H at each occurrence; X 7 is
  • X 1 is
  • X 3 is —(CH 2 ) 2-6 —NH—; n is 1; X 4 is H; X 6 is independently at each occurrence selected from H and —CH 3 ; X 7 is
  • X 1 is
  • X 3 is —(CH 2 ) 2-6 —NH—; n is 1, and X 4 is H.
  • X 1 is
  • X 3 is —(CH 2 ) 2-6 —NH—; n is 1, and X 4 is H.
  • X 1 is
  • X 3 is —(CH 2 ) 2-6 —NH—; n is 1; X 4 is H; X 6 is independently at each occurrence selected from H and —CH 3 , and X 7 is
  • X 3 is —(CH 2 ) 2-6 —NH—; n is 1, and X 4 is H.
  • X 1 is
  • X 3 is —(CH 2 ) 2-6 —NH—; n is 1; X 4 is H, and X 5 is
  • X 1 is
  • X 3 is —(CH 2 ) 2-6 —NH—; n is 0; X 4 is phenyl, and X 5 is
  • X 1 is
  • X 3 is —(CH 2 ) 2-6 —NH—; n is 1; X 4 is phenyl, and X 5 is
  • P has the structure
  • X 1 is
  • X 3 is —(CH 2 ) 2-6 —NH—; X 4 is H, and X 5 is
  • P has the structure selected from the group consisting of:
  • the compound has a half life of longer than 7 days in plasma.
  • the compound does not bind to G protein-coupled receptors (GPCRs) other than GLP1R.
  • GPCRs G protein-coupled receptors
  • composition comprising the compound of any of the embodiments described herein.
  • a pharmaceutical dosage form comprising the compound of any of the embodiments described herein.
  • the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a pancreatic cell, a brain cell, a heart cell, a vascular tissue cell, a kidney cell, an adipose tissue cell, a liver cell, or a muscle cell.
  • provided herein is a method of enhancing GLP1R activity in an individual in need thereof comprising administering to the individual an effective amount of the compound of any of the embodiments described herein, the composition described herein, or the dosage form described herein.
  • provided herein is a method of lowering blood glucose levels in an individual in need thereof comprising administering to the individual an effective amount of the compound of any of the embodiments described herein, the composition described herein, or the dosage form described herein.
  • provided herein is a method of lowering body weight in an individual in need thereof comprising administering to the individual an effective amount of the compound of any of the embodiments described herein, the composition described herein, or the dosage form described herein.
  • a method of treating a GLP1R-associated condition in an individual in need thereof comprising administering to the individual an effective amount of the compound of any of the embodiments described herein, the composition described herein, or the dosage form described herein.
  • the GLP1R-associated condition is type II diabetes, obesity, liver disease, coronary artery disease, or kidney disease.
  • the GLP1R-associated condition is type II diabetes and/or obesity.
  • the compound, the composition, or the dosage form of the present disclosure is administered subcutaneously, intravenously, intradermally, intraperitoneally, or intramuscularly.
  • P has the structure selected from the group consisting of:
  • a carbamate group a cyclodextrin; a polyethylene glycol (PEG) segment having 1 to 36 —CH 2 CH 2 O— (EG) units; a —(CH 2 ) 2-24 — chain; a triazole; one or more amino acids selected from glycine, serine, glutamic acid, alanine, valine, and proline, and combinations thereof;
  • PEG polyethylene glycol
  • A is C or N
  • X 2 is selected from
  • a carbamate group a cyclodextrin; a polyethylene glycol (PEG) segment having 1 to 36 —CH 2 CH 2 O— (EG) units; one or more amino acids selected from glycine, serine, glutamic acid, alanine, valine, and proline, and combinations thereof;
  • PEG polyethylene glycol
  • A is C or N
  • the compound described above has a structure selected from the group consisting of:
  • an antibody-drug conjugate comprising a Glucagon-like peptide-1 receptor (GLP1R)-targeting antibody or an antigen-binding fragment thereof conjugated, optionally through a linker, to a payload having the structure selected from the group consisting of: Structure
  • an antibody-drug conjugate comprising a Glucagon-like peptide-1 receptor (GLP1R)-targeting antibody or an antigen-binding fragment thereof and a payload having the structure (SEQ ID NO: 124):
  • the payload has the structure (SEQ ID NO: 90):
  • an antibody-drug conjugate comprising a Glucagon-like peptide-1 receptor (GLP1R)-targeting antibody or an antigen-binding fragment thereof and a linker-payload having the structure (SEQ ID NO: 125):
  • FIG. 1 A shows a schematic representation of an exemplary antibody-tethered drug conjugate (ATDC) design and its mechanism of action.
  • ATDC antibody-tethered drug conjugate
  • FIG. 1 B shows a schematic representation of a conventional antibody-drug conjugate (ADC) design and its mechanism of action.
  • ADC antibody-drug conjugate
  • FIG. 2 shows a model of an antibody-tethered drug conjugate having an antibody binding to extracellular domain (ECD) and a payload binding to the transmembrane domain (TMD).
  • ECD extracellular domain
  • TMD transmembrane domain
  • FIG. 3 A shows a schematic representation of GLP1 (7-36) amide (SEQ ID NO: 4).
  • the numbers above the sequence correspond to the amino acid positions in the proglucagon propeptide.
  • the blue arrow indicates the dipeptidyl peptidase-4 (DPP-IV) cleavage site.
  • the red arrows indicate the neutral endopeptidase (NEP) cleavage sites.
  • the dashed arrows indicate cleavage sites by unknown endoprotease(s).
  • the residues in orange are amino acids which, when substituted, reduce GLP1R binding and cAMP production.
  • the residues in green are amino acids which, when substituted, reduce GLP1R binding.
  • FIG. 3 B shows a structure of GLP1R bound to GLP1 (Protein Data Bank ID: 3IOL). References of this structure may be found in Zhang et al. Nature 2017, Chepurny et al. JBC 2019, De Graaf et al. Pharmacological reviews 2016, and Manandhar and Ahn Journal of Medical Chemistry 2014, each of which is incorporated herein by reference in its entirety.
  • FIG. 4 A shows the sequence and structure of a GLP1 peptidomimetic, Peptide 5 (SEQ ID NO: 5). The numbers above the sequence correspond to the amino acid positions in the proglucagon propeptide.
  • FIG. 4 B shows superimposed structures of GLP1R bound to Peptide 5 (Protein Data Bank ID: 5NX2) and GLP1R bound to GLP1 (Protein Data Bank ID: 3IOL) using the GLP1R in 5NX2 as the template.
  • 5NX2 structure can be found in Jazayeri A, et al. Nature volume 546, pages 254-258 (2017), which is incorporated herein by reference in its entirety.
  • FIG. 5 shows a synthetic scheme for making GLP1 peptidomimetic payloads of the present disclosure. Solid Phase Peptide Synthesis on resin was established which efficiently generated the payloads of the present disclosure with good yields. Additional GLP1R peptidomimetic payloads were generated via systematic R1/R2/R3-modifications.
  • FIGS. 6 A- 6 D demonstrate that the GLP1R peptidomimetic payloads of the present disclosure showed no activation in related GPCRs bioassays.
  • FIGS. 7 A- 7 B show that shorter linker GLP1R ATDCs showed greater potency over the control ATDCs.
  • FIG. 8 shows that the lead linker-payload showed optimal in vitro ADME profile with no in vitro cardiotoxicity and mutagenic potential and its ATDC is highly stable in plasmas.
  • FIG. 9 A shows two methods for conjugating linker-payloads to an antibody of the present disclosure.
  • FIG. 9 B shows a representative hydrophobic interaction chromatography (HIC) graph of anti-GLP1R ATDC drug loading profile.
  • HIC hydrophobic interaction chromatography
  • FIG. 10 shows CRE-dependent luciferase reporter activity by anti-GLP1R ATDCs.
  • Anti-GLP1R ATDCs showed better in vitro potency than isotype control ATDCs.
  • Unconjugated mAbs did not activate hGLP1R cells (not shown).
  • ATDCs did not activate Glucagon-like peptide-2 receptor (GLP2R), glucagon receptor (GCGR), or gastric inhibitory polypeptide receptor (GIPR) (not shown).
  • GLP2R Glucagon-like peptide-2 receptor
  • GCGR glucagon receptor
  • GIPR gastric inhibitory polypeptide receptor
  • FIG. 11 A shows cyclic AMP response element (CRE)-dependent luciferase reporter activity by anti-GLP1R ATDCs in the presence of unconjugated anti-GLP1R antibodies. It shows that the unconjugated anti-GLP1R mAb concentrations ⁇ 10 nM had no impact on anti-GLP1R ATDC activity. 100 nM unconjugated anti-GLP1R mAb reduced anti-GLP1R ATDC potency by 3.8-fold. The assay was performed by adding unconjugated anti-GLP1R mAb first, then immediately adding anti-GLP1R ATDC, and incubating for 4 hours.
  • CRE cyclic AMP response element
  • FIG. 11 B shows the data corresponding to the graphs in FIG. 11 A .
  • FIG. 12 shows a schematic representation of an exemplary GLP1R Q-tag mAb-GLP1R agonist conjugate of the present disclosure.
  • FIG. 13 shows a general synthetic scheme for preparing GLP1 peptidomimetics according to the disclosure.
  • FIG. 14 shows a sequence for solid-supported synthesis of GLP1 peptidomimetic payloads P1 and P8 according to the disclosure.
  • FIG. 15 shows a sequence for solid-supported synthesis of GLP1 peptidomimetic payloads P2 and P9 according to the disclosure.
  • FIG. 16 shows a sequence for solid-supported synthesis of GLP1 peptidomimetic payloads P3, P4, P5, P6, P7, P11, P13, P14, P15, P16 and P17 according to the disclosure.
  • FIGS. 17 A and 17 B show a sequence for solid-supported synthesis of GLP1 peptidomimetic payloads P10, P12, P18, P19, P25, P26, P27, P28, P29, P30, P31, P36, P37, and P38 according to the disclosure.
  • FIG. 18 shows a sequence for solid-supported synthesis of GLP1 peptidomimetic payloads P20 and P21 according to the disclosure.
  • FIG. 20 shows a sequence for solid-supported synthesis of GLP1 peptidomimetic payload P24 according to the disclosure.
  • FIG. 21 shows a sequence for solid-supported synthesis of GLP1 peptidomimetic payloads P32, P33, P34 and P35 according to the disclosure.
  • FIG. 22 shows a sequence for solid-supported synthesis of GLP1 peptidomimetic payload P39 according to the disclosure.
  • FIG. 23 shows a sequence for solid-supported synthesis of GLP1 peptidomimetic payload P40 according to the disclosure.
  • FIG. 24 shows a sequence for solid-supported synthesis of GLP1 peptidomimetic payload P41 according to the disclosure.
  • FIG. 25 shows a sequence for solid-supported synthesis of GLP1 peptidomimetic payload P42 according to the disclosure.
  • FIG. 26 shows a synthetic route for preparation of Linker-Payloads LP1, LP2, LP3, LP4 and LP5 according to the disclosure.
  • FIG. 27 shows a synthetic route for preparation of Linker-Payloads LP6 and LP7 according to the disclosure.
  • FIG. 28 shows a synthetic route for preparation of Linker-Payloads LP8, LP9, LP10 and LP11 according to the disclosure.
  • FIG. 29 shows a synthetic route for preparation of Linker-Payload LP12 according to the disclosure.
  • FIG. 30 shows a synthetic route for preparation of Linker-Payloads LP13 and LP14 according to the disclosure.
  • FIG. 31 shows a synthetic route for preparation of Linker-Payloads LP15 and LP18 according to the disclosure.
  • FIG. 32 shows a synthetic route for preparation of Linker-Payload LP17 according to the disclosure.
  • FIG. 33 shows a synthetic route for preparation of Linker-Payloads LP18 and LP20 according to the disclosure.
  • FIG. 34 shows a synthetic route for preparation of Linker-Payload LP19 according to the disclosure.
  • FIG. 35 shows a synthetic route for preparation of Linker-Payload LP21 according to the disclosure.
  • FIG. 36 shows a synthetic route for preparation of Linker-Payload LP22 according to the disclosure.
  • FIG. 37 shows a synthetic route for preparation of Linker-Payload LP23 according to the disclosure.
  • FIG. 38 shows a synthetic route for preparation of Linker-Payload LP24 according to the disclosure.
  • FIG. 39 shows a synthetic route for preparation of Linker-Payload LP25 according to the disclosure.
  • FIG. 40 shows a synthetic route for preparation of Linker-Payload LP26 according to the disclosure.
  • FIG. 41 shows a synthetic route for preparation of Linker-Payloads LP27 and LP28 according to the disclosure.
  • FIG. 42 shows a synthetic route for preparation of Linker-Payload LP29 according to the disclosure.
  • FIG. 43 shows a synthetic route for preparation of Linker-Payload LP30 according to the disclosure.
  • FIG. 44 shows a synthetic route for preparation of Linker-Payload LP31 according to the disclosure.
  • FIG. 45 shows a synthetic route for preparation of Linker-Payload LP32 according to the disclosure.
  • FIG. 46 shows a synthetic route for preparation of Linker-Payload LP33 according to the disclosure.
  • FIG. 47 shows a synthetic route for preparation of Linker-Payload LP34 according to the disclosure.
  • FIG. 48 shows a synthetic route for preparation of Linker-Payload LP35 according to the disclosure.
  • FIG. 49 shows a synthetic route for preparation of Linker-Payloads LP36, LP37, LP38, LP39, LP40, and LP41 according to the disclosure.
  • FIG. 50 shows a synthetic route for preparation of Linker-Payload LP42 according to the disclosure.
  • FIG. 51 shows a synthetic route for preparation of Linker-Payload LP43 according to the disclosure.
  • FIG. 52 shows a synthetic route for preparation of Linker-Payload LP44 according to the disclosure.
  • FIG. 53 shows a synthetic route for preparation of Linker-Payload LP45 according to the disclosure.
  • FIG. 54 shows a schematic of a general two-step conjugation procedure for the preparation of site-specific antibody-drug conjugates.
  • FIG. 55 shows a schematic of a general one-step conjugation procedure for the preparation of site-specific antibody-drug conjugates.
  • FIG. 56 shows the commander voltage protocol for electrophysiological study. From a holding potential of ⁇ 80 mV, the voltage was first stepped to ⁇ 50 mV for 80 ms for leak subtraction, and then stepped to +20 mV for 4800 ms to open hERG channels. After that, the voltage was stepped back down to ⁇ 50 mV for 5000 ms, causing a “rebound” or tail current, which was measured and collected for data analysis. Finally, the voltage was stepped back to the holding potential ( ⁇ 80 mV, 1000 ms). Voltage command protocol was repeated every 20 sec and performed continuously during the test (vehicle control and test compound).
  • FIG. 57 shows in vitro stability of anti-GLP1R mAB2-LP11 over a 7-day, 37° C. incubation in mouse, monkey and human plasma.
  • FIG. 58 shows the effects of GLP1R ATDCs on percent body weight changes in obese GLP1R humanized mice.
  • FIG. 59 shows the effects of GLP1R ATDCs on blood glucose levels in obese GLP1R humanized mice.
  • FIG. 60 is a schematic representation of a GLP1R ATDC according to an exemplary embodiment of the disclosure.
  • the present disclosure provides, in some aspects, antibody-drug conjugates that specifically bind the glucagon-like peptide 1 receptor (GLP1R) protein.
  • GLP1R glucagon-like peptide 1 receptor
  • GLP1R and its ligand GLP1 are highly validated targets for obesity and type 2 diabetes.
  • no direct agonist antibodies have been identified for type 2 diabetes treatment.
  • Single peptides with agonist activities on GLP1R are effective therapeutic agents for glucose control and body weight loss, but in-line peptide-antibody fusions are susceptible to proteolysis.
  • antibody-drug conjugates were generated that combine an antibody, or antigen-binding fragment thereof, specifically targeting the extracellular domain of GLP1R, with a GLP1 peptidomimetic functionally activating GLP1R.
  • antibody-drug conjugates of the present disclosure have a longer drug duration with comparable or better weight and glucose reducing efficacy and minimized off-target side effects.
  • treatment comprises methods wherein cells are ablated in such manner where disease is indirectly impacted.
  • treatment comprises depleting immune cells as a hematopoietic conditioning regimen prior to therapy.
  • the subject is a human.
  • the term “effective” applied to dose or amount refers to that quantity of a compound or pharmaceutical composition that is sufficient to result in a desired activity upon administration to a subject in need thereof. Note that when a combination of active ingredients is administered, the effective amount of the combination may or may not include amounts of each ingredient that would have been effective if administered individually. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, the particular drug or drugs employed, the mode of administration, and the like.
  • compositions of the disclosure refers to any salt suitable for administration to a patient.
  • Suitable salts include, but are not limited to, those disclosed in. Berge et al., “Pharmaceutical Salts”, J. Pharm. Sci., 1977, 66:1, incorporated herein by reference.
  • salts include, but are not limited to, acid derived, base derived, organic, inorganic, amine, and alkali or alkaline earth metal salts, including but not limited to calcium salts, magnesium salts, potassium salts, sodium salts, salts of hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methane sulfonic acid, ethane sulfonic acid, p toluene sulfonic acid, salicylic acid, and the like.
  • a payload described herein comprises a tertiary amine, where the nitrogen atom in the tertiary amine is the atom through which the payload is bonded to a linker or a linker-spacer.
  • bonding to the tertiary amine of the payload yields a quaternary amine in the linker-payload molecule.
  • the positive charge on the quaternary amine can be balanced by a counter ion (e.g., chloro, bromo, iodo, or any other suitably charged moiety such as those described herein).
  • Ranges can be expressed herein as from “about” or “approximately” one particular value and/or to “about” or “approximately” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value.
  • alkyl is given its ordinary meaning in the art and may include saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups.
  • a straight chain or branched chain alkyl has about 1-20 carbon atoms in its backbone (e.g., C 1 -C 20 for straight chain, C 2 -C 20 for branched chain), and alternatively, about 1-10 carbon atoms, or about 1 to 6 carbon atoms.
  • a cycloalkyl ring has from about 3-10 carbon atoms in their ring structure where such rings are monocyclic or bicyclic, and alternatively about 5, 6 or 7 carbons in the ring structure.
  • an alkyl group may be a lower alkyl group, wherein a lower alkyl group comprises 1-4 carbon atoms (e.g., C 1 -C 4 for straight chain lower alkyls).
  • alkenyl refers to an alkyl group, as defined herein, having one or more double bonds.
  • alkynyl refers to an alkyl group, as defined herein, having one or more triple bonds.
  • heteroatom means one or more of oxygen, sulfur, nitrogen, phosphorus, or silicon (including, any oxidized form of nitrogen, sulfur, phosphorus, or silicon; the quaternized form of any basic nitrogen or; a substitutable nitrogen of a heterocyclic ring.
  • halogen means F, Cl, Br, or I; the term “halide” refers to a halogen radical or substituent, namely —F, —Cl, —Br, or —I.
  • adduct e.g., “a Diels-Alder adduct” of the present disclosure encompasses any moiety comprising the product of an addition reaction, e.g., a Diels-Alder reaction, independent of the synthetic steps taken to produce the moiety.
  • covalent attachment means formation of a covalent bond, i.e., a chemical bond that involves sharing of one or more electron pairs between two atoms.
  • Covalent bonding may include different interactions, including but not limited to ⁇ -bonding, ⁇ -bonding, metal-to-metal bonding, agostic interactions, bent bonds, and three-center two-electron bonds.
  • first group is said to be “capable of covalently attaching” to a second group, this means that the first group is capable of forming a covalent bond with the second group, directly or indirectly, e.g., through the use of a catalyst or under specific reaction conditions.
  • Non-limiting examples of groups capable of covalently attaching to each other may include, e.g., an amine and a carboxylic acid (forming an amide bond), a diene and a dienophile (via a Diels-Alder reaction), a maleimide and a thiol (forming a thio-maleimide), and an azide and an alkyne (forming a triazole via a 1,3-cycloaddition reaction).
  • compounds of the disclosure may contain “optionally substituted” moieties.
  • substituted whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent.
  • an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position.
  • Combinations of substituents envisioned by this disclosure are preferably those that result in the formation of stable or chemically feasible compounds.
  • stable refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein.
  • structures depicted herein are also meant to include all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational)) forms of the structure; for example, the R and S configurations for each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers. Therefore, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the present compounds are within the scope of the disclosure.
  • cyclic adducts e.g., products of a cycloaddition reaction, e.g., an azide-acetylene cycloaddition reaction or a Diels-Alder reaction
  • cyclic adducts include all regioisomers, i.e., structural isomers that differ only in the position of a functional group or a substituent.
  • the following structures represent triazole regioisomers, which differ only in the position of the substituent on the triazole ring:
  • Triazole regioisomers may also be represented by the following structure:
  • structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms.
  • compounds having the present structures except for the replacement of hydrogen by deuterium or tritium, or the replacement of a carbon by a 11 C- or 13 C- or 14 C-enriched carbon are within the scope of this disclosure.
  • crystalline forms of the compounds of the disclosure and salts thereof are also within the scope of the disclosure.
  • the compounds of the disclosure may be isolated in various amorphous and crystalline forms, including without limitation forms which are anhydrous, hydrated, non-solvated, or solvated.
  • Example hydrates include hemihydrates, monohydrates, dihydrates, and the like.
  • the compounds of the disclosure are anhydrous and non-solvated.
  • anhydrous is meant that the crystalline form of the compound contains essentially no bound water in the crystal lattice structure, i.e., the compound does not form a crystalline hydrate.
  • crystalline form is meant to refer to a certain lattice configuration of a crystalline substance. Different crystalline forms of the same substance typically have different crystalline lattices (e.g., unit cells) which are attributed to different physical properties that are characteristic of each of the crystalline forms. In some instances, different lattice configurations have different water or solvent content.
  • the different crystalline lattices can be identified by solid state characterization methods such as by X-ray powder diffraction (PXRD). Other characterization methods such as differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), dynamic vapor sorption (DVS), solid state NMR, and the like further help identify the crystalline form as well as help determine stability and solvent/water content.
  • Crystalline forms of a substance include both solvated (e.g., hydrated) and non-solvated (e.g., anhydrous) forms.
  • a hydrated form is a crystalline form that includes water in the crystalline lattice. Hydrated forms can be stoichiometric hydrates, where the water is present in the lattice in a certain water/molecule ratio such as for hemihydrates, monohydrates, dihydrates, etc. Hydrated forms can also be non-stoichiometric, where the water content is variable and dependent on external conditions such as humidity.
  • the compounds of the disclosure are substantially isolated.
  • substantially isolated is meant that a particular compound is at least partially isolated from impurities.
  • a compound of the disclosure comprises less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 2.5%, less than about 1%, or less than about 0.5% of impurities.
  • Impurities generally include anything that is not the substantially isolated compound including, for example, other crystalline forms and other substances.
  • a wavy line can intersect or cap a bond or bonds.
  • the wavy line indicates the atom through which the groups, moieties, substituents, or atoms are bonded.
  • GLP1R refers to the glucagon-like peptide 1 receptor and includes recombinant GLP1R protein or a fragment thereof. GLP1R has a sequence of 463 residues. Donnelly, Br J Pharmacol, 166(1):27-41 (2011). Glucagon-like peptide 1 (GLP1) is a 31-amino acid peptide hormone released from intestinal L cells following nutrient consumption.
  • GLP1 The binding of GLP1 to GLP1R potentiates glucose-induced secretion of insulin from pancreatic beta cells, increases insulin expression, inhibits beta-cell apoptosis, promotes beta-cell neogenesis, reduces glucagon secretion, delays gastric emptying, promotes satiety and increases peripheral glucose disposal.
  • an antibody that binds GLP1R or an “anti-GLP1R antibody” includes antibodies and antigen-binding fragments thereof that specifically recognize GLP1R.
  • an “agonist antibody,” as used herein is intended to refer to an antibody whose binding to GLP1R results in activation of at least one biological activity of GLP1R.
  • an agonist antibody of GLP1R may elicit stimulation of the adenylate cyclase pathway resulting in increased synthesis of cyclic AMP and release of insulin if the cell is a mammalian pancreatic beta cell.
  • An agonist antibody of GLP1R may also reduce glucose levels upon administration to a subject in need thereof.
  • protein means any amino acid polymer having more than about 20 amino acids covalently linked via amide bonds.
  • protein includes biotherapeutic proteins, recombinant proteins used in research or therapy, trap proteins and other Fc-fusion proteins, chimeric proteins, antibodies, monoclonal antibodies, human antibodies, bispecific antibodies, antibody fragments, nanobodies, recombinant antibody chimeras, scFv fusion proteins, cytokines, chemokines, peptide hormones, and the like.
  • Proteins can be produced using recombinant cell-based production systems, such as the insect bacculovirus system, yeast systems (e.g, Pichia sp.), mammalian systems (e.g., CHO cells and CHO derivatives like CHO-K1 cells).
  • yeast systems e.g, Pichia sp.
  • mammalian systems e.g., CHO cells and CHO derivatives like CHO-K1 cells.
  • GLP1R means human GLP1R unless specified as being from a non-human species, e.g., “mouse GLP1R,” “monkey GLP1R,” etc.
  • the amino acid sequence of an antibody can be numbered using any known numbering schemes, including those described by Kabat et al., (“Kabat” numbering scheme); Al-Lazikani et al., 1997 , J. Mol. Biol., 273:927-948 (“Chothia” numbering scheme); MacCallum et al., 1996 , J. Mol. Biol. 262:732-745 (“Contact” numbering scheme); Lefranc et al., Dev. Comp. Immunol., 2003, 27:55-77 (“IMGT” numbering scheme); and Honegge and Pluckthun, J. Mol. Biol., 2001, 309:657-70 (“AHo” numbering scheme).
  • Kabat et al. (“Kabat” numbering scheme); Al-Lazikani et al., 1997 , J. Mol. Biol., 273:927-948 (“Chothia” numbering scheme); MacCallum et al., 1996 , J. Mol.
  • the numbering scheme used herein is the Kabat numbering scheme. However, selection of a numbering scheme is not intended to imply differences in sequences where they do not exist, and one of skill in the art can readily confirm a sequence position by examining the amino acid sequence of one or more antibodies. Unless stated otherwise, the “EU numbering scheme” is generally used when referring to a residue in an antibody heavy chain constant region (e.g., as reported in Kabat et al., supra).
  • glutamine-modified antibody refers to an antibody with at least one covalent linkage from a glutamine side chain to a primary amine compound of the present disclosure.
  • the primary amine compound is linked through an amide linkage on the glutamine side chain.
  • the glutamine is an endogenous glutamine.
  • the glutamine is an endogenous glutamine made reactive by polypeptide engineering (e.g., via amino acid deletion, insertion, substitution, or mutation on the polypeptide).
  • the glutamine is polypeptide engineered with an acyl donor glutamine-containing tag (e.g., glutamine-containing peptide tags, Q-tags or TGase recognition tag).
  • TGase recognition tag refers to a sequence of amino acids comprising an acceptor glutamine residue and that when incorporated into (e.g. appended to) a polypeptide sequence, under suitable conditions, is recognized by a TGase and leads to cross-linking by the TGase through a reaction between an amino acid side chain within the sequence of amino acids and a reaction partner.
  • the recognition tag may be a peptide sequence that is not naturally present in the polypeptide comprising the TGase recognition tag.
  • the TGase recognition tag comprises at least one Gln.
  • the TGase recognition tag comprises an amino acid sequence XXQX, wherein X is any amino acid (e.g., conventional amino acid Leu, Ala, Gly, Ser, Val, Phe, Tyr, His, Arg, Asn, Glu, Asp, Cys, Gin, He, Met, Pro, Thr, Lys, or Trp or nonconventional amino acid).
  • X is any amino acid (e.g., conventional amino acid Leu, Ala, Gly, Ser, Val, Phe, Tyr, His, Arg, Asn, Glu, Asp, Cys, Gin, He, Met, Pro, Thr, Lys, or Trp or nonconventional amino acid).
  • the acyl donor glutamine-containing tag comprises an amino acid sequence selected from the group consisting of LLQGG (SEQ ID NO: 6), LLQG (SEQ ID NO: 7), LSLSQG (SEQ ID NO: 8), GGGLLQGG (SEQ ID NO: 9), GLLQG (SEQ ID NO: 10), LLQ, GSPLAQSHGG (SEQ ID NO: 11), GLLQGGG (SEQ ID NO: 12), GLLQGG (SEQ ID NO: 13), GLLQ (SEQ ID NO: 14), LLQLLQGA (SEQ ID NO: 15), LLQGA (SEQ ID NO: 16), LLQYQGA (SEQ ID NO: 17), LLQGSG (SEQ ID NO: 18), LLQYQG (SEQ ID NO: 19), LLQLLQG (SEQ ID NO: 20), SLLQG (SEQ ID NO: 21), LLQLQ (SEQ ID NO: 22), LLQLLQ (SEQ ID NO
  • antibody means any antigen-binding molecule or molecular complex comprising at least one complementarity determining region (CDR) that specifically binds to or interacts with a particular antigen.
  • CDR complementarity determining region
  • antibody includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as multimers thereof (e.g., IgM).
  • Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region.
  • the heavy chain constant region comprises three domains, CH1, CH2, and CH3.
  • Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region.
  • the light chain constant region comprises one domain (CL1).
  • the VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDRs complementarity determining regions
  • FR framework regions
  • Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the FRs of the antibody can be identical to the human germline sequences, or can be naturally or artificially modified.
  • An amino acid consensus sequence can be defined based on a side-by-side analysis of two or more CDRs.
  • antibody also includes antigen-binding fragments of full antibody molecules.
  • antigen-binding portion of an antibody, “antigen-binding fragment” of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex.
  • Antigen-binding fragments of an antibody can be derived, e.g., from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains.
  • DNA is known and/or is readily available from, e.g., commercial sources, DNA libraries (including, e.g., phage-antibody libraries), or can be synthesized.
  • the DNA can be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and/or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.
  • Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab′)2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide.
  • CDR complementarity determining region
  • engineered molecules such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression “antigen-binding fragment,” as used herein.
  • SMIPs small modular immunopharmaceuticals
  • shark variable IgNAR domains are also encompassed within the expression “antigen-binding fragment,” as used herein.
  • variable domain can be of any size or amino acid composition and will generally comprise at least one CDR which is adjacent to or in frame with one or more framework sequences.
  • VH and VL domains can be situated relative to one another in any suitable arrangement.
  • the variable region can be dimeric and contain VH-VH, VH-VL or VL-VL dimers.
  • the antigen-binding fragment of an antibody can contain a monomeric VH or VL domain.
  • an antigen-binding fragment of an antibody can contain at least one variable domain covalently linked to at least one constant domain.
  • variable and constant domains that can be found within an antigen-binding fragment of an antibody of the present description include: (i) VH-CH1; (ii) VH-CH2; (iii) VH-CH3; (iv) VH-CH1-CH2; (V) VH-CH1-CH2—CH3; (vi) VH-CH2-CH3; (vii) VH-CL; (viii) VL-CH1; (ix) VL-CH2; (x) VL-CH3; (xi) VL-CH1-CH2; (xii) VL-CH1-CH2-CH3; (xiii) VL-CH2-CH3; and (xiv) VL-CL.
  • variable and constant domains can be either directly linked to one another or can be linked by a full or partial hinge or linker region.
  • a hinge region can consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60, or more) amino acids which result in a flexible or semi-flexible linkage between adjacent variable and/or constant domains in a single polypeptide molecule.
  • an antigen-binding fragment of an antibody of the present description can comprise a homo-dimer or hetero-dimer (or other multimer) of any of the variable and constant domain configurations listed herein in non-covalent association with one another and/or with one or more monomeric VH or VL domain (e.g., by disulfide bond(s)).
  • antigen-binding fragments can be monospecific or multispecific (e.g., bispecific).
  • a multispecific antigen-binding fragment of an antibody will typically comprise at least two different variable domains, wherein each variable domain is capable of specifically binding to a separate antigen or to a different epitope on the same antigen.
  • Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be adapted for use in the context of an antigen-binding fragment of an antibody of the present description using routine techniques available in the art.
  • the antibodies of the description are human antibodies.
  • the term “human antibody,” as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences.
  • the human antibodies of the description can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3.
  • the term “human antibody,” as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
  • the antibodies can, in some embodiments, be recombinant human antibodies.
  • the term “recombinant human antibody,” as used herein, is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described further below), antibodies isolated from a recombinant, combinatorial human antibody library (described further below), antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (See, e.g., Taylor et al. (1992) Nucl. Acids Res.
  • Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.
  • an immunoglobulin molecule comprises a stable four chain construct of approximately 150-160 kDa in which the dimers are held together by an interchain heavy chain disulfide bond.
  • the dimers are not linked via inter-chain disulfide bonds and a molecule of about 75-80 kDa is formed composed of a covalently coupled light and heavy chain (half-antibody).
  • the frequency of appearance of the second form in various intact IgG isotypes is due to, but not limited to, structural differences associated with the hinge region isotype of the antibody.
  • a single amino acid substitution in the hinge region of the human IgG4 hinge can significantly reduce the appearance of the second form (Angal et al. (1993) Molecular Immunology 30: 105) to levels typically observed using a human IgG1 hinge.
  • the instant description encompasses antibodies having one or more mutations in the hinge, CH2 or CH3 region which can be desirable, for example, in production, to improve the yield of the desired antibody form.
  • the antibodies of the description can be isolated or purified antibodies.
  • An “isolated antibody” or “purified antibody,” as used herein, means an antibody that has been identified and separated and/or recovered from at least one component of its natural environment.
  • an antibody that has been separated or removed from at least one component of an organism, or from a tissue or cell in which the antibody naturally exists or is naturally produced is an “isolated antibody” for purposes of the present description.
  • an antibody that has been purified from at least one component of a reaction or reaction sequence is a “purified antibody” or results from purifying the antibody.
  • An isolated antibody also includes an antibody in situ within a recombinant cell.
  • Isolated antibodies are antibodies that have been subjected to at least one purification or isolation step. According to certain embodiments, an isolated antibody or purified antibody can be substantially free of other cellular material and/or chemicals.
  • the antibodies disclosed herein can comprise one or more amino acid substitutions, insertions and/or deletions in the framework and/or CDR regions of the heavy and light chain variable domains as compared to the corresponding germline sequences from which the antibodies were derived. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available from, for example, public antibody sequence databases.
  • the present description includes antibodies, and antigen-binding fragments thereof, which are derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and/or CDR regions are mutated to the corresponding residue(s) of the germline sequence from which the antibody was derived, or to the corresponding residue(s) of another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are referred to herein collectively as “germline mutations”).
  • germline mutations such sequence changes are referred to herein collectively as “germline mutations”.
  • all of the framework and/or CDR residues within the VH and/or VL domains are mutated back to the residues found in the original germline sequence from which the antibody was derived.
  • only certain residues are mutated back to the original germline sequence, e.g., only the mutated residues found within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only the mutated residues found within CDR1, CDR2 or CDR3.
  • one or more of the framework and/or CDR residue(s) are mutated to the corresponding residue(s) of a different germline sequence (i.e., a germline sequence that is different from the germline sequence from which the antibody was originally derived).
  • the antibodies of the present description can contain any combination of two or more germline mutations within the framework and/or CDR regions, e.g., wherein certain individual residues are mutated to the corresponding residue of a particular germline sequence while certain other residues that differ from the original germline sequence are maintained or are mutated to the corresponding residue of a different germline sequence.
  • antibodies and antigen-binding fragments that contain one or more germline mutations can be easily tested for one or more desired property such as, improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, improved drug-to-antibody ratio (DAR) for antibody-drug conjugates, etc.
  • Antibodies and antigen-binding fragments obtained in this general manner are encompassed within the present description.
  • an antibody heavy chain has an N297 mutation.
  • the antibody is mutated to no longer have an asparagine residue at position 297 according to the EU numbering system as disclosed by Kabat et al.
  • an antibody heavy chain has an N297Q or an N297D mutation.
  • Such an antibody can be prepared by site-directed mutagenesis to remove or disable a glycosylation sequence or by site-directed mutagenesis to insert a glutamine residue at site apart from any interfering glycosylation site or any other interfering structure.
  • Such an antibody also can be isolated from natural or artificial sources.
  • Aglycosylated antibodies also include antibodies comprising a T299 or S298P or other mutations, or combinations of mutations that result in a lack of glycosylation.
  • deglycosylated antibody refers to an antibody in which a saccharide group at is removed to facilitate transglutaminase-mediated conjugation.
  • Saccharides include, but are not limited to, N-linked oligosaccharides.
  • deglycosylation is performed at residue N297.
  • removal of saccharide groups is accomplished enzymatically, included but not limited to via PNGase.
  • epitope refers to an antigenic determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule known as a paratope.
  • a single antigen can have more than one epitope.
  • different antibodies can bind to different areas on an antigen and can have different biological effects.
  • Epitopes can be either conformational or linear.
  • a conformational epitope is produced by spatially juxtaposed amino acids from different segments of the linear polypeptide chain.
  • a linear epitope is one produced by adjacent amino acid residues in a polypeptide chain.
  • an epitope can include moieties of saccharides, phosphoryl groups, or sulfonyl groups on the antigen.
  • conjugated protein or “conjugated antibody” as used herein refers to a protein or an antibody covalently linked to one or more chemical moieties.
  • the chemical moiety can include an amine compound of the present disclosure.
  • Linkers (L) and payloads (P) suitable for use with the present disclosure are described in detail herein.
  • a conjugated antibody comprising a therapeutic moiety is an antibody-drug conjugate (ADC), also referred to as an antibody-payload conjugate, or an antibody-linker-payload conjugate.
  • ADC antibody-drug conjugate
  • DAR Drug-to-Antibody Ratio
  • Linker Antibody Ratio is the average number of reactive primary amine compounds conjugated to a binding agent of the present disclosure.
  • binding agents e.g., antibodies
  • primary amine compounds comprising, e.g., a suitable azide or alkyne.
  • the resulting binding agent which is functionalized with an azide or an alkyne can subsequently react with a therapeutic moiety comprising the corresponding azide or alkyne via the 1,3-cycloaddition reaction.
  • phrases “pharmaceutically acceptable amount” refers to an amount effective or sufficient in treating, reducing, alleviating, or modulating the effects or symptoms of at least one health problem in a subject in need thereof.
  • a pharmaceutically acceptable amount of an antibody or antibody-drug conjugate is an amount effective for modulating a biological target using the antibody or antibody-drug-conjugates provided herein.
  • Suitable pharmaceutically acceptable amounts include, but are not limited to, from about 0.001% up to about 10%, and any amount in between, such as about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% of an antibody or antibody-drug-conjugate provided herein.
  • reaction pH refers to the pH of a reaction after all reaction components or reactants have been added.
  • nucleic acid or fragment thereof indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 95%, and more preferably at least about 96%, 97%, 98% or 99% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST or Gap, as discussed below.
  • a nucleic acid molecule having substantial identity to a reference nucleic acid molecule can, in certain instances, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.
  • the term “substantial similarity” or “substantially similar” means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 95% sequence identity, even more preferably at least 98% or 99% sequence identity.
  • residue positions which are not identical differ by conservative amino acid substitutions.
  • a “conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein.
  • Examples of groups of amino acids that have side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartate and glutamate, and (7) sulfur-containing side chains are cysteine and methionine.
  • conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine.
  • a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443-1445.
  • a “moderately conservative” replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix.
  • Sequence similarity for polypeptides is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions.
  • GCG software contains programs such as Gap and Bestfit which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutein thereof. See, e.g., GCG Version 6.1. Polypeptide sequences also can be compared using FASTA using default or recommended parameters, a program in GCG Version 6.1.
  • FASTA e.g., FASTA2 and FASTA3
  • FASTA2 and FASTA3 provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson (2000) supra).
  • Another particular algorithm when comparing a sequence of the description to a database containing a large number of sequences from different organisms is the computer program BLAST, especially BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402.
  • the present disclosure provides protein-drug conjugate compounds, e.g., antibody-drug conjugate compounds, and precursors and intermediates thereof, pharmaceutical compositions, and methods for treating certain diseases in a subject in need of such treatment.
  • the protein-drug conjugate compounds provided herein comprise a binding agent conjugated with a therapeutic moiety, e.g., GLP1 peptidomimetics, as described herein.
  • the present disclosure provides compounds comprising a binding agent according to the present disclosure, (e.g., an antibody or a fragment thereof), conjugated to one or more GLP1 peptidomimetics via non-cleavable linker.
  • a binding agent e.g., an antibody or a fragment thereof
  • Illustrative non-limiting examples include Formula (I) described herein.
  • the binding agent is an antibody, (e.g., a monoclonal antibody)
  • the term “antibody drug conjugate” or ADC is optionally used.
  • antibody drug conjugate or ADC of the disclosure is an antibody-tethered drug conjugate or ATDC.
  • An ATDC is an antibody-drug conjugate wherein the drug is tethered to the antibody by a non-cleavable linker.
  • the non-cleavable linker in an ATDC of the present disclosure is stable after the ATDC is administered into the body, e.g., a human body.
  • the non-cleavable linker can be stable in plasma, e.g., in human plasma, stable upon binding cell surface, or stable upon antibody binding its target antigen and/or GLP1 peptidomimetic binding GLP1R.
  • the non-cleavable linker is more stable in vivo than either the payload or the antibody under the same physiological conditions.
  • an ATDC of the present disclosure may be degraded in the lysosome to release the payload, the linker-payload, and/or its ATDC metabolites/catabolites, which in certain embodiments are effective for GLP1R activation either locally or systematically.
  • the ATDC is stable in plasma and degrades in the lysosome. In some embodiments, the ATDC is stable in plasma and does not degrade in the lysosome.
  • m is 1. In one embodiment, m is an integer from 2 to 4. In one embodiment, m is 2.
  • the present disclosure provides a compound having a structure of Formula (I): BA-L-P (I),
  • the linker L is a non-cleavable linker, i.e., a linker which is stable and provides a covalent connection between the antibody and the drug, e.g., between a GLP1R-targeting antibody and a GLP1 peptidomimetic payload P according to the present disclosure.
  • the non-cleavable linker L of the present disclosure is stable after the ATDC is administered into the body, e.g., a human body.
  • the linker L can be stable in plasma, e.g., in human plasma, stable upon binding cell surface, or stable upon antibody binding its target antigen and/or GLP1 peptidomimetic binding GLP1R.
  • the linker L is more stable in vivo than either the payload or the antibody under the same physiological conditions.
  • the linker L has the structure of formula (L′): —La—Y-Lp- (L′),
  • Y is a group comprising a triazole.
  • Y is a group comprising a Diels-Alder adduct.
  • the linker L has the structure of formula (L′): —La—Y-Lp- (L′),
  • La comprises C 1-6 alkyl, phenyl, —NH—, —C(O)—, —(CH 2 ) u —NH—C(O)—, —(CH 2 ) u —C(O)—NH—, —(CH 2 —CH 2 —O) v —, —(CH 2 ) u —(O—CH 2 —CH 2 ) v —C(O)—NH—, a peptide unit comprising from 2 to 4 amino acids, or combinations thereof, each of which may be optionally substituted with one or more of —S—, —S(O 2 )—, —C(O)—, —C(O 2 )—; and CO 2 H, wherein subscripts u and v are independently an integer from 1 to 8.
  • La is selected from the group consisting of:
  • R A is a group comprising an alkyne, an azide, a tetrazine, a trans-cyclooctene, a maleimide, an amine, a ketone, an aldehyde, a carboxylic acid, an ester, a thiol, a sulfonic acid, a tosylate, a halide, a silane, a cyano group, a carbohydrate group, a biotin group, a lipid residue and wherein subscripts x, n, p and q are independently an integer from 0 to 12, and combinations thereof.
  • —La— is selected from the group consisting of:
  • a residue e.g., a glutamine residue
  • —La— is
  • —La— is selected from the group consisting of:
  • a residue e.g., a glutamine residue
  • La comprises a polyethylene glycol (PEG) segment having 1 to 36 —CH 2 CH 2 O— (EG) units. In some embodiments, the PEG segment comprises 4 EG units, or 8 EG units, or 12 EG units, or 24 EG units. In some embodiments, the PEG segment comprises 8 EG units. In some embodiments, La has a structure selected from the group consisting of
  • La comprises one or more amino acids selected from glycine, threonine, serine, glutamine, glutamic acid, alanine, valine, leucine, and proline and combinations thereof. In some embodiments, La comprises 1 to 10 glycines and 1 to 6 serines. In some embodiments, La comprises 4 glycines and 1 serine.
  • La is selected from the group consisting of Gly-Gly-Gly-Gly-Ser (G 4 S) (SEQ ID NO: 1), Ser-Gly-Gly-Gly-Gly (SG 4 ) (SEQ ID NO: 2), Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly (G 2 S-G 2 S-G 2 ) (SEQ ID NO: 25), and Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Gly (G 4 S-G 4 ) (SEQ ID NO: 3).
  • La comprises a combination of a PEG segment having 1 to 36 EG units and one or more amino acids selected from glycine, threonine, serine, glutamine, glutamic acid, alanine, valine, leucine, and proline and combinations thereof.
  • La is selected from the group consisting of (SEQ ID NOS 37-38, respectively, in order of appearance):
  • La comprises a —(CH 2 ) 2-24 — chain. In some embodiments, La comprises a combination of a —(CH 2 ) 2-24 — chain, a PEG segment having 1 to 36 EG units and one or more amino acids selected from glycine, threonine, serine, glutamine, glutamic acid, alanine, valine, leucine, and proline and combinations thereof. La is selected from the group consisting of (SEQ ID NOS 127-128, respectively, in order of appearance):
  • Y has a structure selected from the group consisting of:
  • the linker L or the first linker La, or the second linker Lp, comprises a polyethylene glycol (PEG) segment having 1 to 36 —CH 2 CH 2 O— (EG) units.
  • PEG polyethylene glycol
  • the PEG segment comprises between 2 and 30 EG units, or between 4 and 24 EG units. In one embodiment, the PEG segment comprises 2 EG units, or 4 EG units, or 6 EG units, or 8 EG units, or 10 EG units, or 12 EG units, or 14 EG units, or 16 EG units, or 18 EG units, or 20 EG units, or 22 EG units, or 24 EG units.
  • the PEG segment comprises 4 EG units. In one embodiment, the PEG segment comprises 8 EG units. In one embodiment, the PEG segment comprises 12 EG units. In one embodiment, the PEG segment comprises 24 EG units.
  • the PEG segment comprises 4 to 8 EG units. In one embodiment, the PEG segment comprises 4 EG units or 8 EG units.
  • La comprises a PEG segment having 3 EG units.
  • Lp comprises a PEG segment having 4 EG units. In one embodiment, Lp comprises a PEG segment having 8 EG units.
  • the Y-Lp has a structure selected from the group consisting of:
  • p is an integer from 1 to 36.
  • the Y-Lp has a structure selected from the group consisting of:
  • p is an integer from 1 to 36.
  • the linker L or the first linker La, or the second linker Lp comprises one or more amino acids selected from glycine, serine, glutamic acid, alanine, valine, and proline and combinations thereof.
  • the linker L or the first linker La, or the second linker Lp comprises from 1 to 10 glycines, or 1 glycine, or 2 glycines, or 3 glycines, or 4 glycines, or 5 glycines, or 6 glycines, or 7 glycines, or 8 glycines, or 9 glycines, or 10 glycines.
  • the linker L or the first linker La, or the second linker Lp comprises from 1 to 6 serines, or 1 serine, or 2 serines, or 3 serines, or 4 serines, or 5 serines, or 6 serines.
  • the linker L or the first linker La, or the second linker Lp comprises 1 to 10 glycines and 1 to 6 serines.
  • the linker L or the first linker La, or the second linker Lp comprises 4 glycines and 1 serine.
  • the linker L or the first linker La, or the second linker Lp is selected from the group consisting of Gly-Gly-Gly-Gly-Ser (G4S) (SEQ ID NO: 1), Ser-Gly-Gly-Gly-Gly (SG4) (SEQ ID NO: 2), and Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (G4S-G4S) (SEQ ID NO: 3).
  • one or more serine residues comprise a carbohydrate group, e.g., a glucose group.
  • the linker L or the first linker La, or the second linker Lp comprises from 1 to 10 glutamic acids and from 1 to 10 glycines.
  • the linker L or the first linker La, or the second linker Lp comprises a combination of a polyethylene glycol (PEG) segment having 1 to 36 —CH 2 CH 2 O— (EG) units and one or more amino acids selected from glycine, serine, glutamic acid, alanine, valine, and proline and combinations thereof.
  • PEG polyethylene glycol
  • the linker L or the first linker La, or the second linker Lp comprises a combination of a PEG segment having 1 to 36 EG units and 1 to 10 glycines.
  • the linker L or the first linker La, or the second linker Lp comprises a combination of a PEG segment having 1 to 36 EG units and a group selected from Gly-Gly-Gly-Gly-Ser (G4S) (SEQ ID NO: 1), Ser-Gly-Gly-Gly-Gly (SG4) (SEQ ID NO: 2), and Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (G4S-G4S) (SEQ ID NO: 3).
  • the linker L or the first linker La, or the second linker Lp has a structure selected from the group consisting of:
  • Y is the group comprising a triazole, e.g., as shown above, and P is the payload, and wherein Rc is selected from hydrogen (H) and glucose, g is an integer from 1 to 10 and s is an integer from 0 to 4.
  • the Y-Lp has a structure selected from the group consisting of (SEQ ID NOS 31-36, respectively, in order of appearance):
  • the linker L comprises a cyclodextrin moiety.
  • the linker L is attached to the antibody or an antigen-binding fragment thereof via a glutamine residue. In some embodiments, the linker L is attached to the antibody or an antigen-binding fragment thereof via a lysine residue. In some embodiments, the linker L is attached to the antibody or an antigen-binding fragment thereof via a cysteine residue.
  • the payloads P according to the present disclosure have a structure of Formula selected from the group consisting of Formula (P-IB), Formula (P-IIB), and Formula (P-IIIB):
  • the payloads P according to the present disclosure have a structure of Formula (II) (SEQ ID NO: 85):
  • the payload P has a structure selected from:
  • the payload has the structure of formula (P-I), shown above, wherein
  • X 3 is selected from —(CH 2 ) 2-6 —NH—and —(CH 2 ) 2-6 —Tr-, where Tr is a triazole moiety; n is 1, and X 4 is H;
  • X 3 is —(CH 2 ) 2-6 —NH—; n is 1; X 4 is H, and X 5 is selected from —OH, —NH 2 , —NH—OH, and
  • X 3 is —(CH 2 ) 2-6 —NH—; n is 1, and X 4 is H;
  • X 3 is —(CH 2 ) 2-6 —NH—; n is 1; X 4 is H; X 6 is independently at each occurrence selected from H and —CH 2 OH, and X 7 is H;
  • X 3 is —(CH 2 ) 2-6 —Tr-, where Tr is a triazole moiety; n is 1; X 4 is H, and X 5 is
  • X 3 is —(CH 2 ) 2-6 —NH—; n is 1; X 4 is H; X 6 is independently at each occurrence selected from H and —CH 3 ; X 7 is
  • X 8 is —NH 2 ;
  • X 3 is —(CH 2 ) 2-6 —NH—; n is 1; X 4 is H, and X 3 is H;
  • X 3 is —(CH 2 ) 2-6 —NH—; n is 1; X 4 is H; X 6 is H at each occurrence; X 7 is
  • X 3 is —(CH 2 ) 2-6 —NH—; n is 1; X 4 is H; X 6 is independently at each occurrence selected from H and —CH 3 ; X 7 is
  • X 3 is —(CH 2 ) 2-6 —NH—; n is 1, and X 4 is H;
  • X 3 is —(CH 2 ) 2-6 —NH—; n is 1; X 4 is H; X 1 is
  • X 3 is —(CH 2 ) 2-6 —NH—; n is 1, and X 4 is H; X 6 is independently at each occurrence selected from H and —CH 3 , and X 7 is
  • X 3 is —(CH 2 ) 2-6 —NH—; n is 1, and X 4 is H;
  • X 3 is —(CH 2 ) 2-6 —NH—; n is 1; X 4 is H, and X 5 is
  • X 3 is —(CH 2 ) 2-6 —NH—; n is 0; X 4 is phenyl, and X 5 is
  • X 3 is —(CH 2 ) 2-6 —NH—; n is 1; X 4 is phenyl, and X 5 is
  • the payload has the structure of formula (P-II), shown above, wherein X 1 is
  • X 3 is —(CH 2 ) 2-6 —NH—; X 4 is H, and X 5 is
  • the payloads P according to the present disclosure have a structure selected from:
  • the payloads as described above have the following properties:
  • the payloads of the present disclosure are amenable to conjugation with a binding agent (e.g., antibody).
  • a binding agent e.g., antibody
  • the present disclosure provides reactive linker-payloads comprising payloads P as described above and linkers capable of covalently attaching to an antibody or an antigen-binding fragment thereof.
  • the linker-payload according to the present disclosure has a structure of Formula (C) (SEQ ID NO: 27):
  • a carbamate group a cyclodextrin; a polyethylene glycol (PEG) segment having 1 to 36 —CH 2 CH 2 O— (EG) units; a —(CH 2 ) 2-24 — chain; a triazole; one or more amino acids selected from glycine, serine, glutamic acid, alanine, valine, and proline, and combinations thereof;
  • PEG polyethylene glycol
  • A is C or N
  • the linker-payload according to the present disclosure has a structure of Formula (III) (SEQ ID NO: 86):
  • a carbamate group a cyclodextrin; a polyethylene glycol (PEG) segment having 1 to 36 —CH 2 CH 2 O— (EG) units; one or more amino acids selected from glycine, serine, glutamic acid, alanine, valine, and proline, and combinations thereof;
  • PEG polyethylene glycol
  • A is C or N
  • the linker-payload LP comprises a cyclodextrin moiety. In some embodiments, the linker-payload LP comprising a cyclodextrin moiety exhibits GLP1R agonism activity.
  • the linker-payloads LP have the structure selected from the group consisting of:
  • LP# Name Structure LP1 (SEQ ID NO: 87) DIBAC-suc- PEG4-P9 LP2 (SEQ ID NO: 87) DIBAC-suc- PEG8-P9 LP3 (SEQ ID NO: 87) DIBAC-suc- PEG12-P9 LP4 (SEQ ID NO: 87) DIBAC-suc- PEG24-P9 LP5 (SEQ ID NO: 87) BCN-PEG4- carbamate-P9 LP6 (SEQ ID NOS 88 and 156, respectively, in order of appearance) DIBAC-suc- G4S-P9 LP7 (SEQ ID NOS 89 and 157, respectively, in order of appearance) DIBAC-suc- SG4-P9 LP8 (SEQ ID NO: 90) DIBAC-suc- PEG4-triazole-P8 LP9 (SEQ ID NO: 90) BCN-PEG4- triazole-P8 LP
  • the linker-payloads as described above have the following properties:
  • the effectiveness of the protein-drug conjugate embodiments described herein depend on the selectivity of the binding agent to bind its binding partner.
  • the binding agent is any molecule capable of binding with some specificity to a given binding partner.
  • the binding agent is within a mammal where the interaction can result in a therapeutic use.
  • the binding agent is in vitro where the interaction can result in a diagnostic use.
  • the binding agent is capable of binding to a cell or cell population.
  • Suitable binding agents of the present disclosure include proteins that bind to a binding partner.
  • Suitable binding agents include, but are not limited to, antibodies, lymphokines, hormones, growth factors, viral receptors, interleukins, or any other cell binding or peptide binding molecules or substances.
  • the binding agent is an antibody.
  • the antibody is selected from monoclonal antibodies, polyclonal antibodies, antibody fragments (Fab, Fab′, and F(ab)2, minibodies, diabodies, tribodies, and the like).
  • Antibodies herein can be humanized using methods described in U.S. Pat. No. 6,596,541 and US Publication No. 2012/0096572, each incorporated by reference in their entirety.
  • BA is a humanized monoclonal antibody.
  • BA can be a monoclonal antibody that binds GLP1R.
  • the antibody can be any antibody deemed suitable to the practitioner of skill.
  • a linker or linker-payload is attached to one or both heavy chains of the antibody or antigen-binding fragment thereof.
  • a linker or linker-payload is attached to one or both heavy chain variable domains of the antibody or antigen-binding fragment thereof.
  • a linker or linker-payload is attached to the N-terminus of one or both heavy chain variable domains of the antibody or antigen-binding fragment thereof. In some embodiments, a linker or linker-payload is attached to the N-terminus of both heavy chain variable domains of the antibody or antigen-binding fragment thereof. In some embodiments, a linker or linker-payload is attached to one or both light chains of the antibody or antigen-binding fragment thereof. In some embodiments, a linker or linker-payload is attached to one or both light chain variable domains of the antibody or antigen-binding fragment thereof.
  • a linker or linker-payload is attached to the N-terminus of one or both light chain variable domains of the antibody or antigen-binding fragment thereof. In some embodiments, a linker or linker-payload is attached to the N-terminus of both light chain variable domains of the antibody or antigen-binding fragment thereof.
  • a linker or linker-payload is attached to the C-terminus of one or both heavy chain variable domains of the antibody or antigen-binding fragment thereof. In some embodiments, a linker or linker-payload is attached to the C-terminus of both heavy chain variable domains of the antibody or antigen-binding fragment thereof. In some embodiments, a linker or linker-payload is attached to one or both light chains of the antibody or antigen-binding fragment thereof. In some embodiments, a linker or linker-payload is attached to one or both light chain variable domains of the antibody or antigen-binding fragment thereof.
  • a linker or linker-payload is attached to the C-terminus of one or both light chain variable domains of the antibody or antigen-binding fragment thereof. In some embodiments, a linker or linker-payload is attached to the C-terminus of both light chain variable domains of the antibody or antigen-binding fragment thereof.
  • the antibody comprises at least one glutamine residue in at least one polypeptide chain sequence. In certain embodiments, the antibody comprises one or more Gln295 residues. In certain embodiments, the antibody comprises two heavy chain polypeptides, each with one Gln295 residue. In further embodiments, the antibody comprises one or more glutamine residues at a site other than a heavy chain 295.
  • Such antibodies can be isolated from natural sources or engineered to comprise one or more glutamine residues. Techniques for engineering glutamine residues into an antibody polypeptide chain are within the skill of the practitioners in the art. In certain embodiments, a glutamine residue is introduced to the N-terminus of an antibody polypeptide chain.
  • a glutamine residue is introduced to the N-terminus of one or both heavy chains of the antibody. In one embodiment, a glutamine residue is introduced to the N-terminus of both heavy chains of the antibody. In another embodiment, the glutamine residue is introduced to the N-terminus of one or both light chains of the antibody. In one embodiment, a glutamine residue is introduced to the N-terminus of both light chains of the antibody. In another embodiment, a glutamine residue is introduced to the N-terminus of one or both heavy chains and one or both light chains of the antibody.
  • a glutamine residue is introduced to the C-terminus of an antibody polypeptide chain. In one embodiment, a glutamine residue is introduced to the C-terminus of one or both heavy chains of the antibody. In one embodiment, a glutamine residue is introduced to the C-terminus of both heavy chains of the antibody. In another embodiment, the glutamine residue is introduced to the C-terminus of one or both light chains of the antibody. In one embodiment, a glutamine residue is introduced to the C-terminus of both light chains of the antibody. In another embodiment, a glutamine residue is introduced to the C-terminus of one or both heavy chains and one or both light chains of the antibody.
  • the antibody or antigen-binding fragment thereof has been modified to comprise a TGase recognition tag.
  • Suitable TGase recognition tags include those described herein.
  • the antibody or antigen-binding fragment thereof has been modified to comprise a Q-tag at the N-terminus of one or both antibody light chains. In certain embodiments, the antibody or antigen-binding fragment thereof has been modified to comprise a Q-tag at the N-terminus of both antibody light chains.
  • the antibody or antigen-binding fragment thereof has been modified to comprise a Q-tag at the N-terminus of one or both antibody heavy chains. In certain embodiments, the antibody or antigen-binding fragment thereof has been modified to comprise a Q-tag at the N-terminus of both antibody heavy chains.
  • the antibody or antigen-binding fragment thereof has been modified to comprise a Q-tag at the C-terminus of one or both antibody light chains. In certain embodiments, the antibody or antigen-binding fragment thereof has been modified to comprise a Q-tag at the C-terminus of both antibody light chains.
  • the antibody or antigen-binding fragment thereof has been modified to comprise a Q-tag at the C-terminus of one or both antibody heavy chains. In certain embodiments, the antibody or antigen-binding fragment thereof has been modified to comprise a Q-tag at the C-terminus of both antibody heavy chains.
  • the antibody or antigen-binding fragment thereof is aglycosylated. In certain embodiments, the antibody antigen-binding fragment thereof is deglycosylated. In certain embodiments, the antibody antigen-binding fragment is a Fab fragment.
  • the antibody can be in any form known to those of skill in the art.
  • the antibody comprises a light chain.
  • the light chain is a kappa light chain.
  • the light chain is a lambda light chain.
  • the antibody comprises a heavy chain.
  • the heavy chain is an IgA.
  • the heavy chain is an IgD.
  • the heavy chain is an IgE.
  • the heavy chain is an IgG.
  • the heavy chain is an IgM.
  • the heavy chain is an IgG1.
  • the heavy chain is an IgG2.
  • the heavy chain is an IgG3.
  • the heavy chain is an IgG4.
  • the heavy chain is an IgA1. In some aspects, the heavy chain is an IgA2.
  • the antibody is an antibody fragment. In some aspects, the antibody fragment is an Fv fragment. In some aspects, the antibody fragment is a Fab fragment. In some aspects, the antibody fragment is a F(ab′)2 fragment. In some aspects, the antibody fragment is a Fab′ fragment. In some aspects, the antibody fragment is an scFv (sFv) fragment. In some aspects, the antibody fragment is an scFv-Fc fragment.
  • the antibody is a monoclonal antibody. In some embodiments, the antibody is a polyclonal antibody.
  • the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is a human antibody.
  • the antibody can have binding specificity for any antigen deemed suitable to those of skill in the art.
  • the antigen is a transmembrane molecule (e.g., receptor) or a growth factor.
  • exemplary antigens include, but are not limited to, molecules such as GLP1R.
  • Some embodiments herein are target specific for therapeutic or diagnostic use.
  • the binding agent is an anti-glucagon-like peptide 1 receptor (GLP1R), i.e., an anti-GLP1R antibody, or an antigen-binding fragment thereof. In some embodiments, the binding agent is an anti-GLP1R agonist antibody, or an antigen-binding fragment thereof.
  • GLP1R anti-glucagon-like peptide 1 receptor
  • suitable anti-GLP1R antibodies are those disclosed in US Publication No. US20060275288A1, which is incorporated herein by reference in its entirety.
  • Exemplary anti-GLP1R antibodies according to the present disclosure include 5A10 and 9A10.
  • suitable anti-GLP1R antibodies include, but not limited to, AB9433-1, h38C2, PA5-111834, NLS1205, MAB2814, EPR21819, and glutazumab.
  • the present disclosure also provides nucleic acid molecules encoding anti-GLP1R antibodies or portions thereof. Also included within the scope of the present disclosure are recombinant expression vectors capable of expressing a polypeptide comprising a heavy or light chain variable region of an anti-GLP1R antibody. For example, the present disclosure includes recombinant expression vectors comprising any of the nucleic acid molecules mentioned above. Further included within the scope of the present disclosure are host cells into which such vectors have been introduced, as well as methods of producing the antibodies or portions thereof by culturing the host cells under conditions permitting production of the antibodies or antibody fragments, and recovering the antibodies and antibody fragments so produced.
  • the present disclosure includes anti-GLP1R antibodies having a modified glycosylation pattern.
  • modification to remove undesirable glycosylation sites may be useful, or an antibody lacking a fucose moiety present on the oligosaccharide chain, for example, to increase antibody dependent cellular cytotoxicity (ADCC) function (see Shield et al. (2002) JBC 277:26733).
  • ADCC antibody dependent cellular cytotoxicity
  • modification of galactosylation can be made in order to modify complement dependent cytotoxicity (CDC).
  • the disclosure provides a pharmaceutical composition comprising a recombinant human antibody or fragment thereof which specifically binds GLP1R and a pharmaceutically acceptable carrier.
  • the disclosure includes a composition which is a combination of an anti-GLP1R antibody and a second therapeutic agent.
  • the second therapeutic agent is any agent that is advantageously combined with an anti-GLP1R antibody. Additional combination therapies and co-formulations involving the anti-GLP1R antibodies of the present disclosure are disclosed elsewhere herein.
  • the disclosure provides therapeutic methods for targeting cells expressing GLP1R using an anti-GLP1R antibody of the disclosure, wherein the therapeutic methods comprise administering a therapeutically effective amount of a pharmaceutical composition comprising an anti-GLP1R antibody of the disclosure to a subject in need thereof.
  • the cell is a mammalian cell.
  • the cell is a human cell.
  • the cell is a pancreatic cell, a brain cell, a heart cell, a vascular tissue cell, a kidney cell, an adipose tissue cell, a liver cell, or a muscle cell.
  • the cell is a pancreatic cell or a brain cell.
  • the anti-GLP1R antibodies (or antigen-binding fragments thereof) can be used to enhance GLP1R activity in the cells.
  • the disclosure provides monospecific anti-GLP1R antibodies for diagnostic applications, such as, e.g., imaging reagents.
  • the disclosure further includes an antibody or antigen-binding fragment that competes for binding to human GLP1R with an antibody described herein.
  • the disclosure furthermore includes an antibody or antigen-binding fragment, wherein the antibody or antigen-binding fragment thereof binds to the same epitope on human GLP1R as an antibody described herein.
  • the present disclosure provides antibody-drug conjugates comprising an anti-GLP1R antibody or antigen-binding fragment thereof as described above and a therapeutic agent (e.g., a GLP1 peptidomimetic).
  • a therapeutic agent e.g., a GLP1 peptidomimetic
  • the antibody or antigen-binding fragment and the payload are covalently attached via a linker, as discussed above.
  • the anti-GLP1R antibody or antigen-binding fragment can be any of the anti-GLP1R antibodies or fragments described herein.
  • the antibody-drug conjugates of the present disclosure are stable in plasma. Plasma stability may be determined using an in vitro or in vivo plasma stability assay, such as those set forth in Example 8.2 or Example 10 below.
  • the antibody-drug conjugates of the present disclosure have a half life of longer than 4 days, longer than 5 days, longer than 6 days, longer than 7 days, longer than 8 days, longer than 9 days, longer than 10 days, longer than 11 days, longer than 12 days, longer than 13 days, longer than 2 weeks, longer than 3 weeks, longer than 4 weeks, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 month, about 3 month, about 4 month, about 5 month, about 6 month, between 5-10 days, between 8-12 days, between 10-15 days, between 12-18 days, between 15-20 days, between 20-30 days, between 1-2 weeks, between 2-3 weeks, between 3-4 weeks, between 4-6 weeks, between 5-8 weeks, between 6-10 weeks, between 1-2 months, between 1.5-3 months, between 2-4 months, between 2.5-5 months, between 3-6 months, or between 4-6 months in plasma.
  • the antibody-drug conjugates of the present disclosure bind to GLP1R with at least a 10-fold greater affinity than other G protein-coupled receptors (GPCRs). In some embodiments, the antibody-drug conjugates of the present disclosure bind to GLP1R with at least a 20-fold, 50-fold, 100-fold, 500-fold, 1000-fold, 10,000-fold greater affinity than other G protein-coupled receptors (GPCRs). In some embodiments, such antibody-drug conjugates exhibit essentially undetectable binding against GPCRs other than GLP1R.
  • Binding of the antibody-drug conjugates to a target molecule can be measured using a standard binding assay available in the relevant art, such as luciferase reporter assay, surface plasmon resonance assay, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, or Western blot assay.
  • a standard binding assay available in the relevant art, such as luciferase reporter assay, surface plasmon resonance assay, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, or Western blot assay.
  • GPCRs other than GLP1R include, but are not limited to, GIPR, GLP2R and GCGR.
  • an antibody drug conjugate of the present disclosure comprises an anti-GLP1R antibody, or antigen-binding fragment thereof, conjugated with a linker payload, wherein the linker payload is attached to the antibody, or antigen-binding fragment thereof, at the N-terminus of a light chain.
  • an antibody drug conjugate of the present disclosure comprises an anti-GLP1R antibody, or antigen-binding fragment thereof, conjugated at the N-terminus of a light chain with a linker payload, wherein the payload has the following structure (SEQ ID NO: 129):
  • an antibody drug conjugate of the present disclosure comprises an anti-GLP1R antibody, or antigen-binding fragment thereof, conjugated with two linker payloads, wherein each linker payload is attached to the antibody, or antigen-binding fragment thereof, at the N-terminus of a light chain.
  • an antibody drug conjugate of the present disclosure comprises an anti-GLP1R antibody, or antigen-binding fragment thereof, conjugated at the N-terminus of each light chain with a linker payload for a total of two linker payloads per each antibody, wherein the payload has the following structure (SEQ ID NO: 129):
  • an antibody-drug conjugate comprising a Glucagon-like peptide-1 receptor (GLP1R)-targeting antibody or an antigen-binding fragment thereof and a payload having the structure (SEQ ID NO: 124):
  • the payload has the structure (SEQ ID NO: 90):
  • an antibody-drug conjugate comprising a Glucagon-like peptide-1 receptor (GLP1R)-targeting antibody or an antigen-binding fragment thereof and a linker-payload having the structure (SEQ ID NO: 125):
  • the linker-payload has the structure (SEQ ID NO: 126):
  • cysteine see, e.g., US 2007/0258987; WO 2013/055993; WO 2013/055990; WO 2013/053873; WO 2013/053872; WO 2011/130598; US 2013/0101546; and U.S. Pat. No. 7,750,116
  • selenocysteine see, e.g., WO 2008/122039; and Hofer et al., Proc. Natl. Acad. Sci ., USA, 2008, 105:12451-12456
  • formyl glycine see, e.g., Carrico et al., Nat. Chem.
  • Lysine conjugation can also proceed through NHS (N-hydroxy succinimide).
  • Linkers can also be conjugated to cysteine residues, including cysteine residues of a cleaved interchain disulfide bond, by forming a carbon bridge between thiols (see, e.g., U.S. Pat. Nos. 9,951,141, 9,950,076).
  • Linkers can also be conjugated to an antigen-binding protein via attachment to carbohydrates (see, e.g., US 2008/0305497, WO 2014/065661, and Ryan et al., Food & Agriculture Immunol., 2001, 13:127-130) and disulfide linkers (see, e.g., WO 2013/085925, WO 2010/010324, WO 2011/018611, and Shaunak et al., Nat. Chem. Biol., 2006, 2:312-313).
  • Site specific conjugation techniques can also be employed to direct conjugation to particular residues of the antibody or antigen binding protein (see, e.g., Schumacher et al.
  • Site specific conjugation techniques include glutamine conjugation via transglutaminase (see e.g., Schibli, Angew Chemie Inter Ed. 2010, 49,9995).
  • Payloads according to the disclosure linked through lysine and/or cysteine, e.g., via a maleimide or amide conjugation, are included within the scope of the present disclosure.
  • the protein-drug conjugates of the present disclosure are produced according to a two-step process, where Step 1 is lysine-based linker conjugation, e.g., with an NHS-ester linker, and Step 2 is a payload conjugation reaction (e.g., a 1,3-cycloaddition reaction).
  • Step 1 is lysine-based linker conjugation, e.g., with an NHS-ester linker
  • Step 2 is a payload conjugation reaction (e.g., a 1,3-cycloaddition reaction).
  • the protein-drug conjugates of the present disclosure are produced according to a two-step process, where Step 1 is cysteine-based linker conjugation, e.g., with a maleimide linker, and Step 2 is a payload conjugation reaction (e.g., a 1,3-cycloaddition reaction).
  • Step 1 cysteine-based linker conjugation, e.g., with a maleimide linker
  • Step 2 is a payload conjugation reaction (e.g., a 1,3-cycloaddition reaction).
  • the protein-drug conjugates of the present disclosure are produced according to a two-step process, where Step 1 is transglutaminase-mediated site specific conjugation and Step 2 is a payload conjugation reaction (e.g., a 1,3-cycloaddition reaction).
  • Step 1 is transglutaminase-mediated site specific conjugation
  • Step 2 is a payload conjugation reaction (e.g., a 1,3-cycloaddition reaction).
  • proteins may be modified in accordance with known methods to provide glutaminyl modified proteins.
  • Techniques for conjugating antibodies and primary amine compounds are known in the art. Site specific conjugation techniques are employed herein to direct conjugation to glutamine using glutamine conjugation via transglutaminase (see e.g., Schibli, Angew Chemie Inter Ed. 2010, 49, 9995).
  • Primary amine-comprising compounds (e.g., linkers La) of the present disclosure can be conjugated to one or more glutamine residues of a binding agent (e.g., a protein, e.g., an antibody, e.g., an anti-GLP1R antibody) via transglutaminase-based chemo-enzymatic conjugation (see, e.g., Dennler et al., Protein Conjugate Chem. 2014, 25, 569-578, and WO 2017/147542).
  • a binding agent e.g., a protein, e.g., an antibody, e.g., an anti-GLP1R antibody
  • transglutaminase-based chemo-enzymatic conjugation see, e.g., Dennler et al., Protein Conjugate Chem. 2014, 25, 569-578, and WO 2017/147542.
  • a binding agent e.g., a protein, e.g., an
  • a binding agent having a glutamine residue (e.g., a Gln295, i.e. Q295 residue) is treated with a primary amine-containing linker La in the presence of the enzyme transglutaminase.
  • the binding agent is aglycosylated. In certain embodiments, the binding agent is deglycosylated.
  • the binding agent (e.g., a protein, e.g., an antibody) comprises at least one glutamine residue in at least one polypeptide chain sequence.
  • the binding agent comprises two heavy chain polypeptides, each with one Gln295 residue.
  • the binding agent comprises one or more glutamine residues at a site other than a heavy chain 295.
  • a binding agent such as an antibody
  • an antibody having a Gln295 residue and/or an N297Q mutation contains one or more additional naturally occurring glutamine residues in their variable regions, which can be accessible to transglutaminase and therefore capable of conjugation to a linker or a linker-payload.
  • An exemplary naturally occurring glutamine residue can be found, e.g., at Q55 of the light chain.
  • the binding agent, e.g., antibody, conjugated via transglutaminase can have a higher than expected LAR value (e.g., a LAR higher than 4). Any such antibodies can be isolated from natural or artificial sources.
  • linkers La according to the present disclosure comprise at least one first reactive group capable of further reaction after transglutamination.
  • the glutaminyl-modified protein e.g., antibody
  • the reactive linker-payload compound Lp-P may comprise a second reactive group that is capable of reacting with the first reactive group of the linker La.
  • a first or second reactive group according to the present disclosure comprises a moiety that is capable of undergoing a 1,3-cycloaddition reaction.
  • the reactive group is an azide.
  • the reactive group comprises an alkyne (e.g., a terminal alkyne, or an internal strained alkyne).
  • the reactive group is compatible with the binding agent and transglutamination reaction conditions.
  • linker La molecules comprise a first reactive group. In certain embodiments of the disclosure, linker La molecules comprise more than one reactive group.
  • the drug-antibody ratio or DAR is from about 1 to about 30, or from about 1 to about 24, or from about 1 to about 20, or from about 1 to about 16, or from about 1 to about 12, or from about 1 to about 10, or from about 1 to about 8, or about 1, 2, 3, 4, 5, 6, 7, or 8 payload molecules per antibody.
  • the DAR is from 1 to 30.
  • the DAR is from 1 to 16.
  • the DAR is from 1 to 8.
  • the DAR is from 1 to 6.
  • the DAR is from 2 to 4.
  • the DAR is from 2 to 3.
  • the DAR is from 0.5 to 3.5.
  • the DAR is about 1, or about 1.5, or about 2, or about 2.5, or about 3, or about 3.5.
  • the DAR is 2.
  • the DAR is 4.
  • the DAR is 8.
  • the present disclosure provides a method of producing the compound having a structure of Formula (A): BA-(L-P) m (A),
  • the present disclosure provides a method of producing the compound having a structure of Formula (A): BA-(L-P) m (A),
  • the present disclosure provides a method of producing a compound having a structure according to Formula (I): BA-L-P (I),
  • the present disclosure provides a method of producing a compound having a structure according to Formula (I): BA-L-P (I),
  • Y is a group comprising a triazole.
  • the glutamine residue Gln is naturally present in a CH2 or CH3 domain of the BA. In another embodiment, the glutamine residue Gln is introduced to the BA by modifying one or more amino acids. In one embodiment, the Gln is Q295 or N297Q.
  • the transglutaminase is microbial transglutaminase (MTG). In one embodiment, the transglutaminase is bacterial transglutaminase (BTG).
  • the compound L-P for use in any of the above methods of producing the compounds of Formula (I) has a structure selected from the group consisting of:
  • the present disclosure provides pharmaceutical compositions comprising the protein-drug conjugates of the present disclosure.
  • compositions comprising a population of protein-drug conjugates according to the present disclosure having a drug-antibody ratio (DAR) of about 0.5 to about 30.0.
  • DAR drug-antibody ratio
  • the composition has a DAR of about 1.0 to about 2.5.
  • the composition has a DAR of about 2.
  • the composition has a DAR of about 3.0 to about 4.5.
  • the composition has a DAR of about 4.
  • the composition has a DAR of about 6.5 to about 8.5.
  • the composition has a DAR of about 8.
  • compositions of the disclosure are formulated with suitable carriers, excipients, and other agents that provide improved transfer, delivery, tolerance, and the like.
  • suitable carriers excipients, and other agents that provide improved transfer, delivery, tolerance, and the like.
  • a multitude of appropriate formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA.
  • formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LIPOFECTINTM, Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax.
  • vesicles such as LIPOFECTINTM, Life Technologies, Carlsbad, CA
  • DNA conjugates such as LIPOFECTINTM, Life Technologies, Carlsbad, CA
  • DNA conjugates such as LIPOFECTINTM, Life Technologies, Carlsbad, CA
  • DNA conjugates such as LIPOFECTINTM, Life Technologies, Carlsbad, CA
  • DNA conjugates such as LIPOFECTINTM, Life Technologies, Carlsbad, CA
  • the dose of a protein-drug conjugate administered to a patient may vary depending upon the age and the size of the patient, target disease, conditions, route of administration, and the like.
  • the suitable dose is typically calculated according to body weight or body surface area.
  • intravenously administer the protein-drug conjugate of the present disclosure normally at a single dose of about 0.01 to about 20 mg/kg body weight, more preferably about 0.02 to about 7, about 0.03 to about 5, or about 0.05 to about 3 mg/kg body weight.
  • the frequency and the duration of the treatment can be adjusted.
  • Effective dosages and schedules for administering a protein-drug conjugate may be determined empirically; for example, patient progress can be monitored by periodic assessment, and the dose adjusted accordingly.
  • interspecies scaling of dosages can be performed using well-known methods in the art (e.g., Mordenti et al., 1991, Pharmaceut. Res. 8:1351).
  • Various delivery systems are known and can be used to administer the pharmaceutical composition of the disclosure, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the mutant viruses, receptor mediated endocytosis (see, e.g., Wu et al., 1987, J. Biol. Chem. 262:4429-4432).
  • Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes.
  • composition may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.
  • epithelial or mucocutaneous linings e.g., oral mucosa, rectal and intestinal mucosa, etc.
  • Administration can be systemic or local.
  • a pharmaceutical composition of the present disclosure can be delivered subcutaneously or intravenously with a standard needle and syringe.
  • a pen delivery device readily has applications in delivering a pharmaceutical composition of the present disclosure.
  • Such a pen delivery device can be reusable or disposable.
  • a reusable pen delivery device generally utilizes a replaceable cartridge that contains a pharmaceutical composition. Once all of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can readily be discarded and replaced with a new cartridge that contains the pharmaceutical composition. The pen delivery device can then be reused.
  • a disposable pen delivery device there is no replaceable cartridge. Rather, the disposable pen delivery device comes prefilled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.
  • Numerous reusable pen and autoinjector delivery devices have applications in the subcutaneous delivery of a pharmaceutical composition of the present disclosure.
  • Examples include, but are not limited to AUTOPENTM (Owen Mumford, Inc., Woodstock, UK), DISETRONICTM pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75/25TM pen, HUMALOGTM pen, HUMALIN 70/30TM pen (Eli Lilly and Co., Indianapolis, IN), NOVOPENTM 1, ⁇ and Ill (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIORTM (Novo Nordisk, Copenhagen, Denmark), BDTM pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPENTM OPTIPEN PROTM, OPTIPEN STARLETTM, and OPTICLIKTM (sanofi-aventis, Frankfurt, Germany), to name only a few.
  • Examples of disposable pen delivery devices having applications in subcutaneous delivery of a pharmaceutical composition of the present disclosure include, but are not limited to the SOLOSTARTM pen (sanofi-aventis), the FLEXPENTM (Novo Nordisk), and the KWIKPENTM (Eli Lilly), the SURECLICKTM Autoinjector (Amgen, Thousand Oaks, CA), the PENLETTM (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey, L. P.), and the HUMIRATM Pen (Abbott Labs, Abbott Park IL), to name only a few.
  • the pharmaceutical composition can be delivered in a controlled release system.
  • a pump may be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201).
  • polymeric materials can be used; see, Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida.
  • a controlled release system can be placed in proximity of the composition's target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other controlled release systems are discussed in the review by Langer, 1990, Science 249:1527-1533.
  • the injectable preparations may include dosage forms for intravenous, subcutaneous, intracutaneous and intramuscular injections, drip infusions, etc. These injectable preparations may be prepared by methods publicly known. For example, the injectable preparations may be prepared, e.g., by dissolving, suspending or emulsifying the antibody or its salt described above in a sterile aqueous medium or an oily medium conventionally used for injections.
  • aqueous medium for injections there are, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, etc., which may be used in combination with an appropriate solubilizing agent such as an alcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol, polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc.
  • an alcohol e.g., ethanol
  • a polyalcohol e.g., propylene glycol, polyethylene glycol
  • a nonionic surfactant e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil
  • oily medium there are employed, e.g., sesame oil, soybean oil, etc., which may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc.
  • a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc.
  • the pharmaceutical compositions for oral or parenteral use described above are prepared into dosage forms in a unit dose suited to fit a dose of the active ingredients.
  • dosage forms in a unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc.
  • the amount of the aforesaid antibody contained is generally about 5 to about 500 mg per dosage form in a unit dose; especially in the form of injection, it is preferred that the aforesaid antibody is contained in about 5 to about 100 mg and in about 10 to about 250 mg for the other dosage forms.
  • the compounds e.g., an antibody-drug conjugate, a linker-payload and/or a payload
  • the compounds are useful, inter alia, for the treatment, prevention and/or amelioration of a disease, disorder or condition in need of such treatment.
  • the present disclosure provides a method of treating a condition in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a compound (e.g., an antibody-drug conjugate, a linker-payload and/or a payload) according to the disclosure, or the composition comprising any compound according to the present disclosure.
  • a compound e.g., an antibody-drug conjugate, a linker-payload and/or a payload
  • the compounds e.g., an antibody-drug conjugate, a linker-payload and/or a payload
  • the compounds are useful for treating any disease or disorder in which stimulation, activation and/or targeting of GLP1R would be beneficial.
  • the anti-GLP1R antibody-drug conjugates of the present disclosure can be used for the treatment, prevention and/or amelioration of any disease or disorder associated with or mediated by GLP1R expression or activity.
  • the compounds (e.g., an antibody-drug conjugate, a linker-payload and/or a payload) disclosed herein are useful for treating a GLP1R-associated condition.
  • the GLP1R-associated condition is Type 1 or Type 2 diabetes mellitus.
  • the administered compound (e.g., an antibody-drug conjugate, a linker-payload and/or a payload) may cause at least one of the following results: induction of insulin secretion, suppression of glucagon release, reduction of blood sugar, improvement of glycemic control, promotion of islet neogenesis, and delay of gastric emptying or potentiation of glucose resistant islets.
  • the GLP1R-associated condition is a neurodegenerative disorder, a cognitive disorder, memory disorder or learning disorder.
  • the neurodegenerative disorder may be, for example, dementia, senile dementia, mild cognitive impairment, Alzheimer-related dementia, Huntington's chores, tardive dyskinesia, hyperkinesias, mania, Morbus Parkinson, steel-Richard syndrome, Down's syndrome, myasthenia gravis, nerve trauma, brain trauma, vascular amyloidosis, cerebral hemorrhage I with amyloidosis, brain inflammation, Friedrich's ataxia, acute confusion disorder, amyotrophic lateral sclerosis, glaucoma and Alzheimer's disease.
  • the GLP1R-associated condition is a liver disease.
  • the liver disease may be, for example, non-alcoholic fatty liver disease (NAFLD), fatty liver, non-alcoholic steatohepatitis (NASH), and cirrhosis.
  • NAFLD non-alcoholic fatty liver disease
  • NASH non-alcoholic steatohepatitis
  • cirrhosis cirrhosis
  • the GLP1R-associated condition is a coronary artery disease.
  • the coronary artery disease may be, for example, cardiomyopathy and myocardial infarction.
  • the GLP1R-associated condition is a kidney disease.
  • the kidney disease may be, for example, hypertension, or chronic kidney failure.
  • the GLP1R-associated condition is an eating disorder.
  • the eating disorder may be, for example, binge eating.
  • the compounds e.g., an antibody-drug conjugate, a linker-payload and/or a payload
  • apoptosis-mediated degenerative diseases of the central nervous system such as Alzheimer's Disease, Creutzfeld-Jakob Disease and bovine spongiform encephalopathy, chronic wasting syndrome and other prion mediated apoptotic neural diseases (see, e.g., Perry and Grieg (2004) Current Drug Targets 6:565-571).
  • a compound e.g., an antibody-drug conjugate, a linker-payload and/or a payload
  • a compound e.g., an antibody-drug conjugate, a linker-payload and/or a payload
  • ⁇ APP e.g., an antibody-drug conjugate, a linker-payload and/or a payload
  • the compounds (e.g., an antibody-drug conjugate, a linker-payload and/or a payload) disclosed herein may be used to improve learning and memory, for example, by enhancing neuronal plasticity and facilitation of cellular differentiation (see, During et al. (2003) Nature Medicine 9:1173-1179). Further, the compounds (e.g., an antibody-drug conjugate, a linker-payload and/or a payload) disclosed herein may also be used to preserve dopamine neurons and motor function in Morbus Parkinson (see, e.g., Greig et al. (2005) Abstract 897.6, Society for Neuroscience, Washington, D.C.).
  • the compounds e.g., an antibody-drug conjugate, a linker-payload and/or a payload
  • a metabolic disorder may be, for example, obesity, dyslipidemia, metabolic syndrome X, and pathologies emanating from islet insufficiency.
  • Additional diseases that may be treated by a compound (e.g., an antibody-drug conjugate, a linker-payload and/or a payload) of the present disclosure include autoimmune diseases, in particular, those associated with inflammation, including, but not limited to, autoimmune diabetes, adult onset diabetes, morbid obesity, Metabolic Syndrome X and dyslipidemia.
  • the anti-GLP1R antibody-drug conjugate can be employed as a growth factor for the promotion of islet growth in persons with autoimmune diabetes.
  • the compounds (e.g., an antibody-drug conjugate, a linker-payload and/or a payload) described herein may also be useful in the treatment of congestive heart failure.
  • the present disclosure provides a method of selectively targeting an antigen (e.g., GLP1R) on a surface of a cell with a compound.
  • the method of selectively targeting an antigen (e.g., GLP1R) on a surface of a cell with a compound comprises linking the compound to a targeted antibody.
  • the compound is a payload as described above.
  • the cell is a mammalian cell.
  • the cell is a human cell.
  • the cell is a pancreatic cell or a brain cell.
  • the present disclosure provides a method of selectively targeting an antigen on a surface of a cell with a compound having the structure selected from the group consisting of:
  • the present disclosure also includes the use of a compound (e.g., an antibody-drug conjugate, a linker-payload and/or a payload) of the present disclosure in the manufacture of a medicament for the treatment of a disease or disorder (e.g., cancer) related to or caused by GLP1R-expressing cells.
  • a disease or disorder e.g., cancer
  • the present disclosure relates to a protein-drug conjugate comprising an anti-GLP1R antibody or antigen-binding fragment, as disclosed herein, for use in medicine.
  • the present disclosure relates to a compound comprising an antibody-drug conjugate (ADC) as disclosed herein, for use in medicine.
  • ADC antibody-drug conjugate
  • the present disclosure provides methods which comprise administering a pharmaceutical composition comprising any of the exemplary protein-drug conjugates (e.g., antibody-drug conjugates), linker-payloads and payloads described herein in combination with one or more additional therapeutic agents.
  • a pharmaceutical composition comprising any of the exemplary protein-drug conjugates (e.g., antibody-drug conjugates), linker-payloads and payloads described herein in combination with one or more additional therapeutic agents.
  • Exemplary additional therapeutic agents that may be combined with or administered in combination with protein-drug conjugates (e.g., antibody-drug conjugates), linker-payloads and payloads of the present disclosure include, other GLP1R agonists (e.g., an anti-GLP1R antibody or a small molecule agonist of GLP1R or an anti-GLP1R antibody-drug conjugate).
  • protein-drug conjugates e.g., antibody-drug conjugates
  • linker-payloads and payloads of the present disclosure include, other GLP1R agonists (e.g., an anti-GLP1R antibody or a small molecule agonist of GLP1R or an anti-GLP1R antibody-drug conjugate).
  • GLP1R agonists include exenatide (Byetta, Bydureon), liraglutide (Victoza, Saxenda), lixisenatide (Lyxumia in Europe, Adlyxin in the United States), albiglutide (Tanzeum), dulaglutide (Trulicity), semaglutide (Ozempic), and taspoglutide.
  • Exemplary additional therapeutic agents may include dual or triple-agonists, including GLP1R/GIPR dual agonists, such as GLP1R/GCGR dual agonists, GLP1R/GIPR/GCGR triple-agonists.
  • agents that may be beneficially administered in combination with the protein-drug conjugates include those that are useful in the treatment of diabetes (e.g., type II diabetes), obesity, and/or other related metabolic diseases.
  • the additional therapeutic agent is an antidiabetic agent.
  • antidiabetic agents include insulin, insulin analogs (including insulin lispro, insulin aspart, insulin glulisine, isophane insulin, insulin zinc, insulin glargine, and insulin detemir), biguanides (including metformin, phenformin, and buformin), thiazolidinediones or TZDs (including rosiglitazone, pioglitazone, and troglitazone), sulfonylureas (including tolbutamide, acetohexamide, tolazamide, chlorpropamide, glipizide, glibenclamide, glimepiride, gliclazide, glyclopyramide, and gliquidone), meglitinides (including repaglinide and nateglinide), alpha-glucosidase inhibitors (including miglitol), alpha-glucosidase inhibitors (including miglitol),
  • the antidiabetic agent is an oral antidiabetic agents (OAA) such as metformin, acarbose, or TZDs. In some such embodiments, the antidiabetic agent is metformin.
  • OOA oral antidiabetic agents
  • the GLP1R agonist and one or more antidiabetic agents may be formulated into the same dosage form, such as a solution or suspension for parenteral administration.
  • the additional therapeutically active component(s) may be administered just prior to, concurrent with, or shortly after the administration of a compound of the present disclosure; (for purposes of the present disclosure, such administration regimens are considered the administration of an antigen-binding molecule “in combination with” an additional therapeutically active component).
  • the present disclosure includes pharmaceutical compositions in which protein-drug conjugates (e.g., antibody-drug conjugates), linker-payloads and/or payloads of the present disclosure are co-formulated with one or more of the additional therapeutically active component(s) as described elsewhere herein.
  • protein-drug conjugates e.g., antibody-drug conjugates
  • linker-payloads and/or payloads of the present disclosure are co-formulated with one or more of the additional therapeutically active component(s) as described elsewhere herein.
  • multiple doses of a protein-drug conjugate e.g., an anti-GLP1R antibody-drug conjugate
  • linker-payload and/or a payload may be administered to a subject over a defined time course.
  • the methods according to this aspect of the disclosure comprise sequentially administering to a subject multiple doses of a protein-drug conjugate (e.g., an anti-GLP1R antibody-drug conjugate), linker-payload and/or a payload of the disclosure.
  • “sequentially administering” means that each dose of a protein-drug conjugate (e.g., an anti-GLP1R antibody-drug conjugate), linker-payload and/or a payload is administered to the subject at a different point in time, e.g., on different days separated by a predetermined interval (e.g., hours, days, weeks or months).
  • a protein-drug conjugate e.g., an anti-GLP1R antibody-drug conjugate
  • linker-payload and/or a payload is administered to the subject at a different point in time, e.g., on different days separated by a predetermined interval (e.g., hours, days, weeks or months).
  • the present disclosure includes methods which comprise sequentially administering to the patient a single initial dose of a protein-drug conjugate (e.g., an anti-GLP1R antibody-drug conjugate), linker-payload and/or a payload, followed by one or more secondary doses of the protein-drug conjugate (e.g., an anti-GLP1R antibody-drug conjugate), linker-payload and/or payload, and optionally followed by one or more tertiary doses of the a protein-drug conjugate (e.g., an anti-GLP1R antibody-drug conjugate), linker-payload and/or payload.
  • a protein-drug conjugate e.g., an anti-GLP1R antibody-drug conjugate
  • linker-payload and/or a payload e.g., an anti-GLP1R antibody-drug conjugate
  • the terms “initial dose,” “secondary doses,” and “tertiary doses,” refer to the temporal sequence of administration of the protein-drug conjugate (e.g., an anti-GLP1R antibody-drug conjugate), linker-payload and/or payload of the disclosure.
  • the “initial dose” is the dose which is administered at the beginning of the treatment regimen (also referred to as the “baseline dose”);
  • the “secondary doses” are the doses which are administered after the initial dose; and the “tertiary doses” are the doses which are administered after the secondary doses.
  • the initial, secondary, and tertiary doses may all contain the same amount of the protein-drug conjugate (e.g., an anti-GLP1R antibody-drug conjugate), linker-payload and/or payload, but generally may differ from one another in terms of frequency of administration. In certain embodiments, however, the amount of the protein-drug conjugate (e.g., an anti-GLP1R antibody-drug conjugate), linker-payload and/or payload contained in the initial, secondary and/or tertiary doses varies from one another (e.g., adjusted up or down as appropriate) during the course of treatment.
  • the amount of the protein-drug conjugate e.g., an anti-GLP1R antibody-drug conjugate
  • linker-payload and/or payload contained in the initial, secondary and/or tertiary doses varies from one another (e.g., adjusted up or down as appropriate) during the course of treatment.
  • two or more (e.g., 2, 3, 4, or 5) doses are administered at the beginning of the treatment regimen as “loading doses” followed by subsequent doses that are administered on a less frequent basis (e.g., “maintenance doses”).
  • each secondary and/or tertiary dose is administered 1 to 26 (e.g., 1, 1%, 2, 2%, 3, 3%, 4, 4%, 5, 5%, 6, 6%, 7, 7%, 8, 8%, 9, 9%, 10, 10%, 11, 11%, 12, 12%, 13, 13%, 14, 14%, 15, 15%, 16, 16%, 17, 17%, 18, 18%, 19, 19%, 20, 20%, 21, 21%, 22, 22%, 23, 23%, 24, 24%, 25, 25%, 26, 26%, or more) weeks after the immediately preceding dose.
  • 1 to 26 e.g., 1, 1%, 2, 2%, 3, 3%, 4, 4%, 5, 5%, 6, 6%, 7, 7%, 8, 8%, 9, 9%, 10, 10%, 11, 11%, 12, 12%, 13, 13%, 14, 14%, 15, 15%, 16, 16%, 17, 17%, 18, 18%, 19, 19%, 20, 20%, 21, 21%, 22, 22%, 23, 23%, 24, 24%, 25, 25%, 26, 26%, or more
  • the immediately preceding dose means, in a sequence of multiple administrations, the dose of a protein-drug conjugate (e.g., an anti-GLP1R antibody-drug conjugate), linker-payload and/or payload which is administered to a patient prior to the administration of the very next dose in the sequence with no intervening doses.
  • a protein-drug conjugate e.g., an anti-GLP1R antibody-drug conjugate
  • linker-payload and/or payload which is administered to a patient prior to the administration of the very next dose in the sequence with no intervening doses.
  • the methods according to this aspect of the disclosure may comprise administering to a patient any number of secondary and/or tertiary doses of a protein-drug conjugate (e.g., an anti-GLP1R antibody-drug conjugate), linker-payload and/or payload.
  • a protein-drug conjugate e.g., an anti-GLP1R antibody-drug conjugate
  • linker-payload e.g., an anti-GLP1R antibody-drug conjugate
  • payload e.g., an anti-GLP1R antibody-drug conjugate
  • only a single secondary dose is administered to the patient.
  • two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondary doses are administered to the patient.
  • two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiary doses are administered to the patient.
  • each secondary dose may be administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient 1 to 2 weeks after the immediately preceding dose.
  • each tertiary dose may be administered at the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the patient 2 to 4 weeks after the immediately preceding dose.
  • the frequency at which the secondary and/or tertiary doses are administered to a patient can vary over the course of the treatment regimen. The frequency of administration may also be adjusted during the course of treatment by a physician depending on the needs of the individual patient following clinical examination.
  • Term aa# (e.g., aa1) amino acid number (e.g., amino acid 1)
  • Scheme 1 depicts an assembly of peptidomimetic payloads according to the disclosure on resin.
  • the peptides were assembled manually by a roller-mixer onto Fmoc SPPS (Solid phase peptide synthesis) using polypropylene columns equipped with a filter disc.
  • Fmoc SPPS Solid phase peptide synthesis
  • a sufficient quantity of Rink amide MBHA resin (loading: 0.5-0.6 mmol/g) was swollen in DMF or CH 2 Cl 2 for 15 min
  • Step 1 General Procedure for Removal of Fmoc from Fmoc-Rink Amide MBHA Resin
  • the Fmoc-group on the resin was removed by incubation of resin with 20% piperidine in DMF (10-30 ml/100 mg of resin) for 5 to 15 min.
  • the deprotected resin was filtered and washed with excess of DMF and DCM. After washing three times, the resin was incubated in a freshly distilled DMF (1 mL/100 mg of resin), under nitrogen atmosphere for 5 min.
  • Step 2 General Procedure for Amide Coupling on Rink Amide MBHA Resin
  • a DMF solution containing HATU (1.5-4 eq.), Fmoc-protected amino acid (1.5-5 eq. at 0.5M concentration), and DIPEA (5-10 eq.) were added to the resin.
  • the Fmoc-amino acid 5 eq.
  • HATU 4.5 eq.
  • DIPEA 10 eq.
  • the Fmoc-amino acid 1.5-2 eq.
  • HATU 1.5 eq.
  • DIPEA 5.0 eq.
  • the resin-bound peptidomimetic payloads were subjected to cleavage and deprotection with TFA cocktail as follows.
  • a solution of TFA/water/triisopropylsilane (95:2.5:2.5) (10 mL per 100 mg of peptidyl-resin) was added to peptidyl-resins and the mixture was kept at room temperature. After 2-3 hours, the resin was filtered and rinsed by a cleavage solution. The combined filtrate was treated with cold t-BuOMe to precipitate the peptide. The suspension was centrifuged for 10 min (5000 R). The crude white powder was combined and purified by preparative HPLC.
  • the peptide chain elongation was performed by a number of iterations consisting of deprotection, washing, coupling, and washing procedures, (i.e. the resin was subjected to the reaction conditions for 1 hour each time, and the solution was drained and the resin was re-subjected to fresh reagents each time), as depicted in Scheme 1. Finally, the resulting Fmoc-protected peptidyl-resin was deprotected by 20% piperidine as described above and washed with DMF and DCM four times each. The resin bound peptide was dried under nitrogen flow for 10-15 minutes and subjected to cleavage/deprotection.
  • the peptidomimetics designed in the present disclosure were prepared, using Fmoc-SPPS approach. Furthermore, the resin bound peptidomimetics were cleaved and deprotected, purified and characterized using the following protocol.
  • the preparative HPLC was carried out on a Shimadzu LC-8a Liquid chromatograph.
  • a solution of crude peptide dissolved in DMF or water was injected into a column and eluted with a linear gradient of ACN in water. Different methods were used. (See General Information).
  • the desired product eluted were in fractions and the pure peptidomimetics were obtained as amorphous with powders by lyophilization of respective HPLC fractions.
  • the overall recovery was found to be in the range of 40 ⁇ 50% yield.
  • Preparative HPLC method A using FA condition (column: Xtimate C18 150*25 mm*5 ⁇ m; mobile phase: [water (0.225% FA)-ACN]; B %: 40%-70%, 7 min) to afford a pure product.
  • Preparative HPLC method B using TFA condition (column: YMC-Exphere C18 10 ⁇ m 300*50 mm 12 nm; mobile phase: [water (0.1% TFA)-ACN]; B %: 15%-45%, 55 min) to afford a pure product.
  • Preparative HPLC method C using neutral condition (column: Phenomenex Gemini-NX 150*30 mm*5 ⁇ m; mobile phase: [water (10 mM NH 4 HCO 3 )-ACN]; B %: 21%-51%, 11 min) to afford a pure product.
  • Preparative HPLC method D using neutral condition (column: Waters Xbridge 150*255u; mobile phase: [water (10 mM NH 4 HCO 3 )-ACN]; B %: 20%-50%, 7 min) to afford a pure product.
  • Preparative HPLC method E using FA condition (column: Phenomenex Luna C18 250*50 mm*10 ⁇ m; mobile phase: [water (0.225% FA)-ACN]; B %: 55%-86%, 21 min) to afford a pure product.
  • FA condition column: Phenomenex Luna C18 250*50 mm*10 ⁇ m; mobile phase: [water (0.225% FA)-ACN]; B %: 55%-86%, 21 min
  • each peptide was analyzed by analytical HPLC with using methods A, B, C, D, E, or F.
  • the acquisition of chromatogram was carried out at 220 nm, using a PDA detector, in general, the purity of pure peptidomimetics obtained after Prep-HPLC purification was found to be >95%.
  • HPLC method A (20 min): Mobile Phase: 4.0 mL TFA in 4 L water (solvent A) and 3.2 mL TFA in 4 L acetonitrile (solvent B), using the elution gradient 10%-80% (solvent B) over 20 minutes and holding at 80% for 3.5 minutes at a flow rate of 1.0 mL/minutes; Column: Gemini-NX 5 ⁇ m 150*4.6 mm, C18, 110A Wavelength: UV 220 nm, 254 nm; Column temperature: 30° C.
  • HPLC method B 15 min: Mobile Phase: 2.75 mL/4L TFA in water (solvent A) and 2.5 mL/4L TFA in acetonitrile (solvent B), using the elution gradient 10%-80% (solvent B) over 10 minutes and holding at 80% for 5 minutes at a flow rate of 1.5 mL/min; Column: WELCH Ultimate LP-C18 150*4.6 mm 5 ⁇ m; Wavelength: UV 220 nm, 215 nm, 254 nm; Column temperature: 40° C.
  • HPLC method D 15 min: Mobile Phase: water containing 0.04% TFA (solvent A). and acetonitrile containing 0.02% TFA (solvent B), using the elution gradient 10% to 80% (solvent B) over 15 minutes and holding at 80% for 3.5 minutes at a flow rate of 1.5 mL/minutes; Column: YMC-Pack ODS-A 150*4.6 mm Wavelength: UV 220 nm, 254 nm; Column temperature: 30° C.
  • HPLC method E 8 min: Mobile Phase: 0.2 mL/1L NH3*H 2 O in water (solvent A) and acetonitrile (solvent B), using the elution gradient 0%-60% (solvent B) over 5 minutes and holding at 60% for 2 minutes at a flow rate of 1.2 ml/min; Column: Xbridge Shield RP-18, 5 ⁇ m, 2.1*50 mm. Wavelength: UV 220 nm, 254 nm; Column temperature: 30° C.
  • HPLC method F (7 min): Mobile Phase: 1.5 mL/4L TFA in water (solvent A) and 0.75 mL/4L TFA in acetonitrile (solvent B), using the elution gradient 10%-80% (solvent B).
  • Each peptide was characterized by electrospray ionization mass spectrometry (ESI-MS), either in flow injection or LC/MS mode. In all cases, the experimentally measured molecular weight was within 0.5 Daltons of the calculated monoisotopic molecular weight.
  • ESI-MS electrospray ionization mass spectrometry
  • LC-MS method A a MERCK (RP-18e 25-2 mm) column, with a flow rate of 1.5 mL/min, eluting with a gradient of 5% to 95% acetonitrile containing 0.02% TFA (solvent B) and water containing 0.04% TFA (solvent A).
  • LC-MS method B a Xtimate (C18 2.1*30 mm, 3 ⁇ m) column, with a flow rate of 0.8 mL/min, eluting with a gradient of 10% to 80% acetonitrile containing 0.02% TFA (solvent B) and water containing 0.04% TFA (solvent A).
  • LC-MS method C a Chromolith (Flash RP-18e 25-3 mm) column, with a flow rate of 1.5 mL/min, eluting with a gradient of 5% to 95% acetonitrile containing 0.04% TFA (solvent B) and water containing 0.06% TFA (solvent A).
  • LC-MS method D Agilent, a Pursuit (5 C18 20*2.0 mm) column, flow rate 1.5 mL/min, eluting with a gradient of 5% to 95% acetonitrile containing 0.02% TFA (solvent B) and water containing 0.04% TFA (solvent A).
  • LC-MS method E Waters Xbridge C18 30*2.0 mm, 3.5 ⁇ m column, with a flow rate of 1.0 mL/min, eluting with a gradient of 5% to 95%.
  • Mobile phase A) 0.05% N H3 H 2 O in Water; B) ACN.
  • LC-MS method F XBridge C18 3.5 ⁇ m 2.1*30 mm Column, with a flow rate of 1.0 mL/min, Mobile phase: 0.8 mL/4L NH 3 ⁇ H 2 O in water (solvent A) and acetonitrile (solvent B), using the gradient 10%-80% (solvent B) over 2 minutes and holding at 80% for 0.48 minutes.
  • the mobile phase 0.1% FA in water (solvent A) and ACN (solvent B); Elution Gradient: 5%-95% (solvent B) over 3 minutes and holding at 95% for 1 minute at a flow rate of 1 ml/minute; Column: Xbridge Shield RP 18 5 ⁇ m, 2.1*50 mm
  • Ion Source AJS ESI source; Ion Mode: Positive; Nebulization Gas: Nitrogen; Drying Gas (N2) Flow: 8 L/min; Nebulizer Pressure: 35 psig; Gas Temperature: 325° C.; Sheath gas Temperature: 350° C.; Sheath gas flow: 11 L/min; Capillary Voltage: 3.5 KV; Fragmentor Voltage: 175 V.
  • Aa1b-2 were prepared according to the detailed synthetic procedure found in Wu, X. Y.; Stockdill, J. L.; Park, P. K.; Samuel J. Danishefsky, S. J. Expanding the Limits of Isonitrile-Mediated Amidations: On the Remarkable Stereosubtleties of Macrolactam Formation from Synthetic Seco-Cyclosporins. J. Am. Chem. Soc. 2012, 134, 2378-2384. Preparation of aa1b-1 was referred to at Du, J. J.; Gao, X. F.; Xin, L. M.; Lei, Z.; Liu, Z.; and Guo, J. Convergent Synthesis of N-Linked Glycopeptides via Aminolysis of w-Asp p-Nitrophenyl Thioesters in Solution. Org. Lett. 2016, 18, 4828-4831.
  • Step 1 Synthesis of (S,Z)-methyl 5-(4-(benzyloxy)phenyl)-2-((tert-butoxycarbonyl)amino)pent-4-enoate (aa1b-3)
  • Step 2 Synthesis of (S)-methyl 2-((tert-butoxycarbonyl)amino)-5-(4-hydroxyphenyl) pentanoate (aa1b-4)
  • Step 3 Synthesis of (S)-methyl 2-((tert-butoxycarbonyl)amino)-5-(4-(4-chlorobutoxy)phenyl) pentanoate (aa1b-5)
  • Step 4 Synthesis of (S)-methyl 5-(4-(4-azidobutoxy)phenyl)-2-((tert-butoxycarbonyl)amino) pentanoate (aa1b-6)
  • Step 5 Synthesis of (S)-5-(4-(4-azidobutoxy)phenyl)-2-((tert-butoxycarbonyl)amino)pentanoic acid (aa1b-7)
  • Step 6 Synthesis of (S)-2-amino-5-(4-(4-azidobutoxy)phenyl)pentanoic acid hydrochloride (aa1b-8)
  • aa1b-7 (7.25 g, 17.84 mmol, 1.0 eq.) in 4M HCl/EtOAc (75 mL) was stirred at 22° C. for 1 hr. The reaction was concentrated in vacuum to give aa1b-8 (5.2 g, 15.17 mmol, 85.04% yield, HCl) as a white solid.
  • Step 7 Synthesis of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(4-(4-azidobutoxy) phenyl)pentanoic acid (aa1b-9)
  • Step 8 Synthesis of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(4-(4-((tert-butoxycarbonyl) amino) butoxy)phenyl)pentanoic acid (aa1b)
  • the compound aa2-5 was prepared according to the following literature reference: I. Berezowska, N. N. Chung, C. Lemieux, B. C. Wilkes, and P. W. Schiller, Agonist vs Antagonist Behavior of 5 Opioid Peptides Containing Novel Phenylalanine Analogues in Place of Tyr. J. Med. Chem., Vol. 52, No. 21, 2009, 6941-6945.
  • Step 2 Synthesis of 1-bromo-4-((4-methoxybenzyl)oxy)-2-vinylbenzene (aa2-3)
  • Step 3 Synthesis of 2-(4-((4-methoxybenzyl)oxy)-2-vinylphenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (aa2-4)
  • Step 4 Synthesis of (S)-methyl 2-((tert-butoxycarbonyl)amino)-3-(4′-((4-methoxybenzyl)oxy)-2′-vinyl-[1, 1′-biphenyl]-4-yl)propanoate (aa2-6)
  • Step 5 Synthesis of (S)-methyl 2-((tert-butoxycarbonyl)amino)-3-(2′-ethyl-4′-hydroxy-[1,1′-biphenyl]-4-yl)propanoate (aa2-7)
  • Step 7 Synthesis of (S)-methyl 3-(4′-(4-azidobutoxy)-2′-ethyl-[1,1′-biphenyl]-4-yl)-2-((tert-butoxycarbonyl)amino)propanoate (aa2-9)
  • Step 8 Synthesis of (S)-3-(4′-(4-azidobutoxy)-2′-ethyl-[1,1′-biphenyl]-4-yl)-2-((tert-butoxycarbonyl)amino)propanoic acid (aa2-10)
  • Step 9 Synthesis of (S)-2-amino-3-(4′-(4-azidobutoxy)-2′-ethyl-[1,1′-biphenyl]-4-yl)propanoic acid hydrochloride (aa2-11)
  • Step 10 Synthesis of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4′-(4-azidobutoxy)-2′-ethyl-[1, 1′-biphenyl]-4-yl)propanoic acid (aa2)
  • Step 1 Synthesis of (R)-2-((((9H-fluoren-9-y) methoxy) carbonyl) amino)-3-(4′-(4-((tert-butoxycarbonyl)amino)butoxy)-2′-ethyl-[1, 1′-biphenyl]-4-yl) propanoic acid (aa2b)
  • Reagents and conditions (a) HATU (1.5 eq), DHP (1.0 eq), DIPEA (3.0 eq), DCM, 25° C., 2 h; (b) 10% Pd/C, MeOH, 25° C., 5h.
  • aa13-1 was prepared according to WO2010/052253, the content of which are incorporated by reference herein in their entirety.
  • Step 1 Synthesis of benzyl 2,2-dimethyl-3-oxo-3-(((tetrahydro-2H-pyran-2-yl)oxy)amino) propanoate (aa13-2)
  • Step 2 Synthesis of 2,2-dimethyl-3-oxo-3-(((tetrahydro-2H-pyran-2-yl)oxy)amino)propanoic acid (aa13)
  • the compound aa14-1 was prepared according to US2015/380666.
  • Step 1 Synthesis of methyl 2-(1-benzyl-1H-pyrazol-5-yl)-2-methylpropanoate (aa14-3)
  • Step 2 Synthesis of methyl 2-(1-benzyl-1H-pyrazol-5-yl)-2-methylpropanoate (aa14-3)
  • a mixture of aa14-2 (700 mg, 2.71 mmol, 1 eq) and LiOH ⁇ H 2 O (1 M, 5.42 mL, 2 eq) in THF (5.4 mL) was stirred at 50° C. for 16 hr. The reaction was concentrated in vacuum to remove the THF. The reaction was diluted with water (5 mL) and MTBE (5 mL). The aqueous layer was separated and adjusted to pH 6 with 1N HCl. The organic layer was discarded. The aqueous layer was extracted with EtOAc (5 mL*3). The organic layers were combined and washed with brine (5 mL), dried over anhydrous Na 2 SO 4 , filtered and concentrated in vacuum to give aa14-3 (600 mg, 2.46 mmol, 90.64% yield) as a white solid.
  • Reagents and conditions (a) NaH (1.2 eq), dibromoethane (2 eq.), THF (25 ml), 0-100° C., 16h; (b) 2-(1-trityl-1H-imidazol-5-yl) ethanamine (0.9 eq), DMF (5 ml), 60° C., 16h; (c) LiOH (4 eq.), H 2 O (10 ml), THF (10 ml), 20° C., 16h; (d) SFC separation.
  • Step 1 Synthesis of diethyl 2-(2-bromoethyl)-2-methylmalonate (aa20-2)
  • the reaction mixture was then heated to 100° C. in solvent for 14 hr. TLC indicated that the reactant was consumed, and one major new spot with lower polarity was detected.
  • the residue was poured into 1N HCl to adjust pH 2-4. Then aqueous phase was extracted with ethyl acetate (30 mL*3). The combined organic phase was washed with brine (50 mL), dried over anhydrous Na 2 SO 4 , filtered and concentrated in vacuum.
  • the residue was purified by flash silica gel chromatography (ISCO®; 24 g SepaFlash® Silica Flash Column, Eluent of 0-20% Ethyl acetate/Petroleum ether gradient @ 20 mL/min) for 40 min with 0.8 L solvent.
  • the compound aa20-2 (1.2 g, 4.27 mmol, 74.35% yield) was obtained as a pale yellow liquid.
  • Step 2 Synthesis of ethyl 3-methyl-2-oxo-1-(2-(1-trityl-1H-imidazol-5-yl)ethyl)pyrrolidine-3-carboxylate (aa20-3)
  • the crude product was purified by C-18 reverse phase chromatography (ISCO®; 80 g, C-18 Column, Eluent of 0-100% acetonitrile/H 2 O gradient @ 40 mL/min, 60 min with total volume 2400 mL) to provide a yellow solid.
  • the compound aa20-3 (650 mg, 903.47 ⁇ mol, 31.93% yield, 70.55% purity) was obtained as a yellow solid.
  • Step 3 Synthesis of 3-methyl-2-oxo-1-(2-(1-trityl-1H-imidazol-5-yl)ethyl)pyrrolidine-3-carboxylic acid (aa20-4)
  • the crude was purified by prep-HPLC (column: Phenomenex Synergi Max-RP 250*50 mm*10 um; mobile phase: water (0.225% FA)-ACN; B %: 30%-60%, 60 min) to give aa20-4 (180 mg, 375.34 ⁇ mol, 29.31% yield) as a white solid.
  • Step 4 Synthesis of (R)-3-methyl-2-oxo-1-(2-(1-trityl-1H-imidazol-5-yl)ethyl)pyrrolidine-3-carboxylic acid (aa20) and (S)-3-methyl-2-oxo-1-(2-(1-trityl-1H-imidazol-5-yl)ethyl)pyrrolidine-3-carboxylic acid (aa21)
  • Step 1 Synthesis of 2-(1-trityl-1H-imidazol-4-yl) ethanol (aa22-2)
  • Step 2 Synthesis of 2-(1-trityl-1H-imidazol-4-yl)ethyl 4-methylbenzenesulfonate (aa22-3)
  • Step 3 Synthesis of (R)-1-(2-(1-trityl-1H-imidazol-4-yl)ethyl)pyrrolidine-3-carboxylic acid (aa22)
  • Step 4 Synthesis of methyl (3S)-1-[2-(1-tritylimidazol-4-yl)ethyl]pyrrolidine-3-carboxylate (aa23-1)
  • the aqueous phase was extracted with ethyl acetate (50 mL*2).
  • the combined organic phase was washed with brine (15 mL*2), dried over anhydrous Na 2 SO 4 , filtered and concentrated in vacuum.
  • the residue was purified by flash silica gel chromatography (ISCO®; 20 g SepaFlash® Silica Flash Column, Eluent of 0-100% Ethyl acetate/Petroleum ether then 0-10% MeOH (0.5% TEA additive)/DCM gradient @ 30 mL/min) for 25 min with total volume 1.6 L.
  • Product aa23-1 (420 mg, 856.99 ⁇ mol, 58.12% yield, 95% purity) was obtained as a brown oil.
  • Step 5 Synthesis of methyl (3S)-1-[2-(1-tritylimidazol-4-yl)ethyl]pyrrolidine-3-carboxylate (aa23)
  • Reagents and conditions (a) HCl/dioxane, 0-25° C., 2 h, 100%; (b) CbzCl (1.2 eq.), HOSu (1.3 eq.), DIPEA (2 eq.), DCM, 0-20° C., 12 h, 96%; (c) 3a (2 eq.), K 2 CO 3 (2 eq.), 60° C., 12 h, 56%; (d) LiOH (2.0 eq.), THF, H 2 O, 20° C., 12 h, 94%; (e) H 2 , Pd(OH) 2 , AcOH, MeOH, 20° C., 12h; (f) FmocOSu (1.2 eq.), NaHCO 3 (2.0 eq.), THF, H 2 O, 0-20° C., 12 h, 44%; (g) PhNH 2 (1.1 eq.), DIC (1.0 eq.), HOBt (1.0 e
  • Step 1 Synthesis of methyl (2S)-2-amino-3-[4-(2-ethyl-4-hydroxy-phenyl)phenyl]propanoate (aa27-2)
  • Step 2 Synthesis of methyl (2S)-2-(benzyloxycarbonylamino)-3-[4-(2-ethyl-4-hydroxy-phenyl) phenyl]propanoate (aa27-3)
  • Step 4 Synthesis of (S)-2-(((benzyloxy)carbonyl)amino)-3-(4′-(4-((tert-butoxycarbonyl) amino) butoxy)-2′-ethyl-[1, 1′-biphenyl]-4-yl)propanoic acid (aa27-5)
  • Step 5 Synthesis of (2S)-2-amino-3-[4-[4-[4-(tert-butoxycarbonylamino)butoxy]-2-ethyl-phenyl]phenyl]propanoic acid (aa27-6)
  • Step 6 Synthesis of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4′-(4-((tert-butoxycarbonyl)amino)butoxy)-2′-ethyl-[1, 1′-biphenyl]-4-yl)propanoic acid (aa27-7)
  • Step 7 Synthesis of 9H-fluoren-9-ylmethyl N-[(1S)-2-anilino-1-[[4-[4-[4-(tert-butoxycarbonylamino)butoxy]-2-ethyl-phenyl]phenyl]methyl]-2-oxo-ethyl]carbamate (aa27-8)
  • Step 8 Synthesis of tert-butyl N-[4-[4-[4-[4-[(2S)-2-amino-3-anilino-3-oxo-propyl]phenyl]-3-ethyl-phenoxy]butyl]carbamate (aa27)
  • Reagents and conditions (a) PhNH 2 (1.1 eq.), DIC (1.0 eq.), HOBT(1.0 eq.), DCM, 0-20° C., 1h; (b) piperidine (3.0 eq.), DMF, 20° C., 12 h, 52%; (c) 3a (1 eq.), DIC (1.0 eq.), HOBT (1.0 eq.), DCM, 0-20° C., 1 h, 61%; (d) piperidine (15.0 eq.), DMF, 20° C., 0.5 h, 41%.
  • Step 1 Synthesis of 9H-fluoren-9-ylmethyl N-[(1S)-4-(3,5-dimethylphenyl)-1-(phenylcarbamoyl) butyl]carbamate (aa28-1)
  • Step 3 Synthesis of 9H-fluoren-9-ylmethyl N-[(1S)-1-[[4-[4-[4-(tert-butoxycarbonylamino) butoxy]-2-ethyl-phenyl]phenyl]methyl]-2-[[(1S)-4-(3,5-dimethylphenyl)-1-(phenylcarbamoyl) butyl]amino]-2-oxo-ethyl]carbamate (aa28-3)
  • Step 4 Synthesis of tert-butyl N-[4-[4-[4-[(2S)-2-amino-3-[[(1S)-4-(3,5-dimethylphenyl)-1-(phenylcarbamoyl)butyl]amino]-3-oxo-propyl]phenyl]-3-ethyl-phenoxy]butyl]carbamate (aa28)
  • Reagents and conditions (a) PPh 3 (1.2 eq.), toluene, 110° C., 12 h; b) aa30-2A (1.5 eq.), LiHMDS (4.0 eq.), THF, -20-25° C., 12 h; c) H 2 , Pd/C, MeOH, 25° C., 2 h; d) TEMPO (1.5 eq.), PIDA (2.5 eq.), MeCN/H 2 O (3:2), 25° C., 2 h;
  • Step 1 Synthesis of (3-hydroxypropyl)triphenylphosphonium bromide (aa30-2)
  • LCMS conditions 1.5 ML/4L TFA in water (solvent A) and 0.75 ML/4L TFA in acetonitrile (solvent B), using the elution gradient 5%-95% (solvent B) over 0.7 minutes and holding at 95% for 0.4 minutes at a flow rate of 1.5 mL/min; Column: Agilent Pursult 5 C18 20*2.0 mm Wavelength: UV 220 nm & 254 nm; Column temperature: 50° C.; MS ionization: ESI.
  • aa30-2 (10.67 g, 26.60 mmol, 1.5 eq.) in THF (50 mL) was added LiHMDS (1 M, 70.92 mL, 4 eq.). The mixture was stirred at 0° C. for 0.5 h, then a solution of aa30-2A (1-tritylimidazole-4-carbaldehyde, 6 g, 17.73 mmol, 1.0 eq.) in THF (60 mL) was added to the above mixture and the resulting mixture was stirred at 25° C. for 12 h. The reaction progress was monitored by LCMS.
  • LCMS conditions 1.5 ML/4L TFA in water (solvent A) and 0.75 ML/4L TFA in acetonitrile (solvent B), using the elution gradient 5%-95% (solvent B) over 0.7 minutes and holding at 95% for 0.4 minutes at a flow rate of 1.5 mL/min; Column: Agilent Pursult 5 C18 20*2.0 mm Wavelength: UV 220 nm & 254 nm; Column temperature: 50° C.; MS ionization: ESI.
  • Step 3 Synthesis of 4-(1-trityl-1H-imidazol-4-yl)butan-1-ol (aa30-4)
  • LCMS conditions 1.5 ML/4L TFA in water (solvent A) and 0.75 ML/4L TFA in acetonitrile (solvent B), using the elution gradient 5%-95% (solvent B) over 0.7 minutes and holding at 95% for 0.4 minutes at a flow rate of 1.5 mL/min; Column: Agilent Pursult 5 C18 20*2.0 mm Wavelength: UV 220 nm & 254 nm; Column temperature: 50° C.; MS ionization: ESI.
  • Reagents and conditions (a) PPh 3 (1.2 eq.), toluene, 110° C., 12 h; b) aa31-2A (1.5 eq.), tBuOK (3.0 eq.), THF, 0-25° C., 12 h; c) H 2 , Pd/C, MeOH, 25° C., 2 h;
  • Step 1 Synthesis of (3-carboxypropyl)triphenylphosphonium bromide (aa31-2)
  • LCMS conditions 1.5 ML/4L TFA in water (solvent A) and 0.75 ML/4L TFA in acetonitrile (solvent B), using the elution gradient 10%-80% (solvent B) over 1.35 minutes and holding at 80% for 0.9 minutes at a flow rate of 0.8 ml/min; Column: Xtimate C18 2.1*30 mm, 3 mm; Wavelength: UV 220 nm & 254 nm Column temperature: 50° C.; MS ionization: ESI.
  • Step 2 Synthesis of (E)-5-(1-trityl-1H-imidazol-4-yl)pent-4-enoic acid (aa31-3)
  • aa31-2 (3.81 g, 8.87 mmol, 1.5 eq.) in THF (20 mL) was added tBuOK (1.99 g, 17.73 mmol, 3 eq.) at 0° C. under N 2 , the mixture was stirred at 0° C. for 30 min, then a solution of aa31-2A (2 g, 5.91 mmol, 1 eq.) in THF (20 mL) was added to the above mixture at 0° C. and the final mixture was stirred at 25° C. for 12 h. The reaction progress was monitored by LCMS.
  • LCMS conditions 1.5 ML/4L TFA in water (solvent A) and acetonitrile (solvent B), using the elution gradient 10%-80% (solvent B) over 1.35 minutes and holding at 80% for 0.9 minutes at a flow rate of 0.8 ml/min; Column: Xtimate C18 2.1*30 mm, 3 mm; Wavelength: UV 220 nm & 254 nm; Column temperature: 50° C.; MS ionization: ESI.
  • Reagents and conditions (a) PPh 3 (1.2 eq.), toluene, 110° C., 12 h; b) aa32-2A (1.5 eq.), tBuOK (3.0 eq.), THF, 0-25° C., 12 h; c) H 2 , Pd/C, MeOH, 25° C., 2 h;
  • LCMS conditions 1.5 ML/4L TFA in water (solvent A) and 0.75 ML/4L TFA in acetonitrile (solvent B), using the elution gradient 10%-80% (solvent B) over 1.35 minutes and holding at 80% for 0.9 minutes at a flow rate of 0.8 ml/min; Column: Xtimate C18 2.1*30 mm, 3 mm; Wavelength: UV 220 nm & 254 nm Column temperature: 50° C.;
  • aa32-2 (7.86 g, 17.73 mmol, 1.5 eq.) in THF (60 mL) was added t-BuOK (3.98 g, 35.46 mmol, 3 eq.) at 0° C. under N 2 . The mixture was stirred at 0° C. for 30 min. A solution of aa32-2A (4 g, 11.82 mmol, 1 eq.) in THF (40 mL) was added to the above mixture at 0° C. and the resulting mixture was stirred at 25° C. for 12 h. The reaction progress was monitored by LCMS.
  • LCMS conditions 1.5 ML/4L TFA in water (solvent A) and acetonitrile (solvent B), using the elution gradient 10%-80% (solvent B) over 1.35 minutes and holding at 80% for 0.9 minutes at a flow rate of 0.8 ml/min; Column: Xtimate C18 2.1*30 mm, 3 mm; Wavelength: UV 220 nm & 254 nm; Column temperature: 50° C.; MS ionization: ESI.
  • LCMS conditions 1.5 ML/4L TFA in water (solvent A) and 0.75 ML/4L TFA in acetonitrile (solvent B), using the elution gradient 10%-80% (solvent B) over 6.0 minutes and holding at 80% for 0.5 minutes at a flow rate of 0.8 ml/min; Column: Xtimate C18 2.1*30 mm, 3 mm; Wavelength: UV 220 nm & 254 nm Column temperature: 50° C.; MS ionization: ESI.
  • Reagents and conditions (a) PPh 3 (1.2 eq.), toluene, 110° C., 12 h; b) aa33-2A (1.5 eq.), tBuOK (3.0 eq.), THF, 0-25° C., 12 h; c) H 2 , Pd/C, MeOH, 25° C., 2 h;

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